Anti-tnf antibodies, compositions, and methods for the treatment of active ankylosing spondylitis

ABSTRACT

The present invention relates to compositions and methods utilizing anti-TNF antibodies having a heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37 for use in the safe and effective treatment of active Ankylosing Spondylitis (AS).

FIELD OF THE INVENTION

The present invention relates to compositions and methods utilizinganti-TNF antibodies having a heavy chain (HC) comprising SEQ ID NO:36and a light chain (LC) comprising SEQ ID NO:37 for use in the safe andeffective treatment of active Ankylosing Spondylitis (AS).

BACKGROUND OF THE INVENTION

TNF alpha is a soluble homotrimer of 17 kD protein subunits. Amembrane-bound 26 kD precursor form of TNF also exists.

Cells other than monocytes or macrophages also produce TNF alpha. Forexample, human non-monocytic tumor cell lines produce TNF alpha and CD4+and CD8+ peripheral blood T lymphocytes and some cultured T and B celllines also produce TNF alpha.

TNF alpha causes pro-inflammatory actions which result in tissue injury,such as degradation of cartilage and bone, induction of adhesionmolecules, inducing procoagulant activity on vascular endothelial cells,increasing the adherence of neutrophils and lymphocytes, and stimulatingthe release of platelet activating factor from macrophages, neutrophilsand vascular endothelial cells.

TNF alpha has been associated with infections, immune disorders,neoplastic pathologies, autoimmune pathologies and graft-versus-hostpathologies. The association of TNF alpha with cancer and infectiouspathologies is often related to the host's catabolic state. Cancerpatients suffer from weight loss, usually associated with anorexia.

The extensive wasting which is associated with cancer, and otherdiseases, is known as “cachexia”. Cachexia includes progressive weightloss, anorexia, and persistent erosion of lean body mass in response toa malignant growth. The cachectic state causes much cancer morbidity andmortality. There is evidence that TNF alpha is involved in cachexia incancer, infectious pathology, and other catabolic states.

TNF alpha is believed to play a central role in gram-negative sepsis andendotoxic shock, including fever, malaise, anorexia, and cachexia.Endotoxin strongly activates monocyte/macrophage production andsecretion of TNF alpha and other cytokines. TNF alpha and othermonocyte-derived cytokines mediate the metabolic and neurohormonalresponses to endotoxin. Endotoxin administration to human volunteersproduces acute illness with flu-like symptoms including fever,tachycardia, increased metabolic rate and stress hormone release.Circulating TNF alpha increases in patients suffering from Gram-negativesepsis.

Thus, TNF alpha has been implicated in inflammatory diseases, autoimmunediseases, viral, bacterial and parasitic infections, malignancies,and/or neurodegenerative diseases and is a useful target for specificbiological therapy in diseases, such as rheumatoid arthritis and Crohn'sdisease. Beneficial effects in open-label trials with a chimericmonoclonal antibody to TNF alpha (cA2) have been reported withsuppression of inflammation and with successful retreatment afterrelapse in rheumatoid arthritis and in Crohn's disease. Beneficialresults in a randomized, double-blind, placebo-controlled trial with cA2have also been reported in rheumatoid arthritis with suppression ofinflammation.

Other investigators have described mAbs specific for recombinant humanTNF which had neutralizing activity in vitro. Some of these mAbs wereused to map epitopes of human TNF and develop enzyme immunoassays and toassist in the purification of recombinant TNF. However, these studies donot provide a basis for producing TNF neutralizing antibodies that canbe used for in vivo diagnostic or therapeutic uses in humans, due toimmunogenicity, low specificity and/or pharmaceutical unsuitability.

Neutralizing antisera or mAbs to TNF have been shown in mammals otherthan man to abrogate adverse phaysiological changes and prevent deathafter lethal challenge in experimental endotoxemia and bacteremia. Thiseffect has been demonstrated, e.g., in rodent lethality assays and inprimate pathology model systems.

Putative receptor binding loci of hTNF has been disclosed and thereceptor binding loci of TNF alpha as consisting of amino acids 11-13,37-42, 49-57 and 155-157 of TNF have been disclosed.

Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/ormonoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestionor fusion protein products thereof) are potential therapeutic agentsthat are being investigated in some cases to attempt to treat certaindiseases. However, such antibodies or fragments can elicit an immuneresponse when administered to humans. Such an immune response can resultin an immune complex-mediated clearance of the antibodies or fragmentsfrom the circulation, and make repeated administration unsuitable fortherapy, thereby reducing the therapeutic benefit to the patient andlimiting the readministration of the antibody or fragment. For example,repeated administration of antibodies or fragments comprising non-humanportions can lead to serum sickness and/or anaphylaxis. In order toavoid these and other problems, a number of approaches have been takento reduce the immunogenicity of such antibodies and portions thereof,including chimerization and humanization, as well known in the art.These and other approaches, however, still can result in antibodies orfragments having some immunogenicity, low affinity, low avidity, or withproblems in cell culture, scale up, production, and/or low yields. Thus,such antibodies or fragments can be less than ideally suited formanufacture or use as therapeutic proteins.

Accordingly, there is a need to provide anti-TNF antibodies or fragmentsthat overcome one more of these problems, as well as improvements overknown antibodies or fragments thereof.

SUMMARY OF THE INVENTION

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion, and wherein apatient treated with the anti-TNF antibody achieves an ASDAS inactivedisease (<1.3) at 4 weeks of treatment or at 2 weeks of treatment.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion at a dose of 2mg/kg over 30±10 minutes at Weeks 0 and 4, and then every 8 weeks (q8w)thereafter, and wherein a patient treated with the anti-TNF antibodyachieves an ASDAS inactive disease (<1.3) at 4 weeks of treatment or at2 weeks of treatment.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said antibody isadministered with or without methotrexate (MTX), sulfasalazine (SSZ) orhydroxychloroquine (HCQ) and wherein said anti-TNF antibody isadministered via intravenous (IV) infusion, and wherein a patienttreated with the anti-TNF antibody achieves an ASDAS inactive disease(<1.3) at 4 weeks of treatment or at 2 weeks of treatment.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered via IV infusion, and wherein apatient treated with the composition achieves an ASDAS inactive disease(<1.3) at 4 weeks of treatment or at 2 weeks of treatment.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered via IV infusion at a dose of 2mg/kg over 30±10 minutes at Weeks 0 and 4, then every 8 weeks (q8w)thereafter, and wherein a patient treated with the composition achievesan ASDAS inactive disease (<1.3) at 4 weeks of treatment or at 2 weeksof treatment.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered with or without methotrexate(MTX), sulfasalazine (SSZ) or hydroxychloroquine (HCQ) and wherein saidcomposition is administered via IV, and wherein a patient treated withthe composition achieves an ASDAS inactive disease (<1.3) at 4 weeks oftreatment or at 2 weeks of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion, and wherein a patient treated with thecomposition achieves an ASDAS inactive disease (<1.3) at 4 weeks oftreatment or at 2 weeks of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion at a dose of 2 mg/kg over 30±10 minutes atWeeks 0 and 4, then every 8 weeks (q8w) thereafter, and wherein apatient treated with the composition achieves an ASDAS inactive disease(<1.3) at 4 weeks of treatment or at 2 weeks of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered with or without methotrexate (MTX), sulfasalazine (SSZ) orhydroxychloroquine (HCQ), and wherein said composition is administeredvia IV infusion at a dose of 2 mg/kg over 30±10 minutes at Weeks 0 and4, then every 8 weeks (q8w) thereafter, and wherein a patient treatedwith the composition achieves an ASDAS inactive disease (<1.3) at 4weeks of treatment or at 2 weeks of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion, and wherein a patient treated with thecomposition achieves an ASDAS inactive disease (<1.3) at 4 weeks oftreatment or at 2 weeks of treatment, the method further comprisingadministering, prior, concurrently or after said (a) administering, atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion, and wherein atweek 16 of treatment patients treated with the anti-TNF antibody achievea mean change from baseline in one or more criteria selected from thegroup consisting of: Bath Ankylosing Spondylitis Functional Index(BASFI)=−2.4±2.1 standard deviation (SD), Bath Ankylosing SpondylitisMetrology Index (BASMI)=−0.4±0.6 SD, 36-item Short-Form Health SurveyPhysical Component Summary (SF-36 PCS)=8.5±7.5 SD, 36-item Short-FormHealth Survey Mental Component Summary (SF-36 MCS)=6.5±9.1 SD, andAnkylosing Spondylitis Qualify of Life (ASQoL)=−5.4±5.0 SD.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion at a dose of 2mg/kg over 30±10 minutes at Weeks 0 and 4, and then every 8 weeks (q8w)thereafter, and wherein at week 16 of treatment patients treated withthe anti-TNF antibody achieve a mean change from baseline in one or morecriteria selected from the group consisting of: BASFI=−2.4±2.1 SD,BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, andASQoL=−5.4±5.0 SD.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered with or without methotrexate (MTX),sulfasalazine (SSZ) or hydroxychloroquine (HCQ) and the anti-TNFantibody is administered via intravenous (IV) infusion at a dose of 2mg/kg, and wherein at week 16 of treatment patients treated with theanti-TNF antibody achieve a mean change from baseline in one or morecriteria selected from the group consisting of: BASFI=−2.4±2.1 SD,BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, andASQoL=−5.4±5.0 SD.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered via IV infusion, and wherein atweek 16 of treatment patients treated with the anti-TNF antibody achievea mean change from baseline in one or more criteria selected from thegroup consisting of: BASFI=−2.4±2.1 SD, BASMI=−0.4±0.6 SD, SF-36PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, and ASQoL=−5.4±5.0 SD.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered via IV infusion at a dose of 2mg/kg over 30±10 minutes at Weeks 0 and 4, then every 8 weeks (q8w)thereafter, and wherein at week 16 of treatment patients treated withthe anti-TNF antibody achieve a mean change from baseline in one or morecriteria selected from the group consisting of: BASFI=−2.4±2.1 SD,BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, andASQoL=−5.4±5.0 SD.

The present invention provides a composition comprising at least oneisolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,and at least one pharmaceutically acceptable carrier or diluent for usein the safe and effective treatment of active Ankylosing Spondylitis,wherein said composition is administered with or without methotrexate(MTX), sulfasalazine (SSZ) or hydroxychloroquine (HCQ) and thecomposition is administered via IV infusion, and wherein at week 16 oftreatment patients treated with the anti-TNF antibody achieve a meanchange from baseline in one or more criteria selected from the groupconsisting of: BASFI=−2.4±2.1 SD, BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5SD, SF-36 MCS=6.5±9.1 SD, and ASQoL=−5.4±5.0 SD.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion, and wherein at week 16 of treatmentpatients treated with the anti-TNF antibody achieve a mean change frombaseline in one or more criteria selected from the group consisting of:BASFI=−2.4±2.1 SD, BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36MCS=6.5±9.1 SD, and ASQoL=−5.4±5.0 SD.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion at a dose of 2 mg/kg over 30±10 minutes atWeeks 0 and 4, then every 8 weeks (q8w) thereafter, and wherein at week16 of treatment patients treated with the anti-TNF antibody achieve amean change from baseline in one or more criteria selected from thegroup consisting of: BASFI=−2.4±2.1 SD, BASMI=−0.4±0.6 SD, SF-36PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, and ASQoL=−5.4±5.0 SD.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered with or without methotrexate (MTX), sulfasalazine (SSZ) orhydroxychloroquine (HCQ), and wherein the composition is administeredvia IV infusion, and wherein at week 16 of treatment patients treatedwith the anti-TNF antibody achieve a mean change from baseline in one ormore criteria selected from the group consisting of: BASFI=−2.4±2.1 SD,BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36 MCS=6.5±9.1 SD, andASQoL=−5.4±5.0 SD.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising: administering a compositioncomprising a safe and effective amount of an isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37, wherein said composition isadministered via IV infusion, and wherein at week 16 of treatmentpatients treated with the anti-TNF antibody achieve a mean change frombaseline in one or more criteria selected from the group consisting of:BASFI=−2.4±2.1 SD, BASMI=−0.4±0.6 SD, SF-36 PCS=8.5±7.5 SD, SF-36MCS=6.5±9.1 SD, and ASQoL=−5.4±5.0 SDA, the method further comprisingadministering, prior, concurrently or after said (a) administering, atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment or prevention of Ankylosing Spondylitis, wherein said anti-TNFantibody is administered to a subject and induces a clinical responseselected from the group consisting of the responses in the table below:

Efficacy at week 16 Placebo Golimumab 2 mg/kg Patients randomized, n 103105 Clinical efficacy ASAS20, n (%) 27 (26.2%) 77 (73.3%)** ASAS40, n(%) 9 (8.7%) 50 (47.6%)** BASDAI 50, n (%) 15 (14.6%) 43 (41.0%)** ASASpartial remission, n (%) 4 (3.9%) 17 (16.2%)*  *p < 0.01; **p ≤ 0.001ASAS20/40, ≥20%/40% improvement in ASsessment in Ankylosing Spondylitis(ASAS) International Working Group criteria; BASDAI, Bath AnkylosingSpondylitis Disease Activity Index

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion, and wherein ≥65%of patients receiving the treatment achieve ASAS20 at week 16 oftreatment.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion, and wherein ≥65%of patients receiving the treatment achieve ASAS20 at week 16 oftreatment with a treatment difference (improvement compared to placebo)of ≥45%.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active Ankylosing Spondylitis, wherein said anti-TNFantibody is administered via intravenous (IV) infusion at a dose of 2mg/kg, administered over 30±10 minutes, at Weeks 0 and 4, and then every8 weeks (q8w) thereafter, and wherein ≥65% of patients receiving thetreatment achieve ASAS20 at week 16 of treatment.

The present invention provides at least one isolated mammalian anti-TNFantibody having a heavy chain (HC) comprising SEQ ID NO:36 and a lightchain (LC) comprising SEQ ID NO:37 for use in the safe and effectivetreatment of active

Ankylosing Spondylitis, wherein said anti-TNF antibody is administeredvia intravenous (IV) infusion with or without methotrexate (MTX),sulfasalazine (SSZ) or hydroxychloroquine (HCQ), and wherein ≥65% ofpatients receiving the treatment achieve ASAS20 at week 16 of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising:

(a) administering a composition comprising a safe and effective amountof an isolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,wherein said composition is administered via IV infusion, and wherein≥65% of patients receiving the treatment achieve an ASAS20 at week 16 oftreatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising:

(a) administering a composition comprising a safe and effective amountof an isolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,wherein said composition is administered via IV infusion, and wherein≥65% of patients receiving the treatment achieve an ASAS20 at week 16 oftreatment with a treatment difference (improvement compared to placebo)of ≥45%.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising:

(a) administering a composition comprising a safe and effective amountof an isolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,wherein said composition is administered via IV infusion at a dose of 2mg/kg, administered over 30±10 minutes, at Weeks 0 and 4, and then every8 weeks (q8w) thereafter, and wherein ≥65% of patients receiving thetreatment achieve an ASAS20 at week 16 of treatment.

The present invention provides a method for treating a TNF relatedcondition, wherein the TNF related condition is active AnkylosingSpondylitis, the method comprising:

(a) administering a composition comprising a safe and effective amountof an isolated mammalian anti-TNF antibody having a heavy chain (HC)comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37,wherein said composition is administered via IV infusion with or withoutmethotrexate (MTX), sulfasalazine (SSZ) or hydroxychloroquine (HCQ), andwherein ≥65% of patients receiving the treatment achieve an ASAS20 atweek 16 of treatment.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a graphical representation showing an assay for ability ofTNV mAbs in hybridoma cell supernatants to inhibit TNFα binding torecombinant TNF receptor. Varying amounts of hybridoma cell supernatantscontaining known amounts of TNV mAb were preincubated with a fixedconcentration (5 ng/ml) of ¹²⁵I-labeled TNFα. The mixture wastransferred to 96-well Optiplates that had been previously coated withp55-sf2, a recombinant TNF receptor/IgG fusion protein. The amount ofTNFα that bound to the p55 receptor in the presence of the mAbs wasdetermined after washing away the unbound material and counting using agamma counter. Although eight TNV mAb samples were tested in theseexperiments, for simplicity three of the mAbs that were shown by DNAsequence analyses to be identical to one of the other TNV mAbs (seeSection 5.2.2) are not shown here. Each sample was tested in duplicate.The results shown are representative of two independent experiments.

FIGS. 2A-B shows DNA sequences of the TNV mAb heavy chain variableregions. The germline gene shown is the DP-46 gene. ‘TNVs’ indicatesthat the sequence shown is the sequence of TNV14, TNV15, TNV148, andTNV196. The first three nucleotides in the TNV sequence define thetranslation initiation Met codon. Dots in the TNV mAb gene sequencesindicate the nucleotide is the same as in the germline sequence. Thefirst 19 nucleotides (underlined) of the TNV sequences correspond to theoligonucleotide used to PCR-amplify the variable region. An amino acidtranslation (single letter abbreviations) starting with the mature mAbis shown only for the germline gene. The three CDR domains in thegermline amino acid translation are marked in bold and underlined. Lineslabeled TNV148(B) indicate that the sequence shown pertains to bothTNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due tothe sequence not being known or not existing in the germline gene. TheTNV mAb heavy chains use the J6 joining region.

FIG. 3 shows DNA sequences of the TNV mAb light chain variable regions.The germline gene shown is a representative member of the Vg/38K familyof human kappa germline variable region genes. Dots in the TNV mAb genesequences indicate the nucleotide is the same as in the germlinesequence. The first 16 nucleotides (underlined) of the TNV sequencescorrespond to the oligonucleotide used to PCR-amplify the variableregion. An amino acid translation of the mature mAb (single letterabbreviations) is shown only for the germline gene. The three CDRdomains in the germline amino acid translation are marked in bold andunderlined. Lines labeled TNV148(B) indicate that the sequence shownpertains to both TNV148 and TNV148B. Gaps in the germline DNA sequence(CDR3) are due to the sequence not being known or not existing in thegermline gene. The TNV mAb light chains use the J3 joining sequence.

FIG. 4 shows deduced amino acid sequences of the TNV mAb heavy chainvariable regions. The amino acid sequences shown (single letterabbreviations) were deduced from DNA sequence determined from bothuncloned PCR products and cloned PCR products. The amino sequences areshown partitioned into the secretory signal sequence (signal), framework(FW), and complementarity determining region (CDR) domains. The aminoacid sequence for the DP-46 germline gene is shown on the top line foreach domain. Dots indicate that the amino acid in the TNV mAb isidentical to the germline gene. TNV148(B) indicates that the sequenceshown pertains to both TNV148 and TNV148B. ‘TNVs’ indicates that thesequence shown pertains to all TNV mAbs unless a different sequence isshown. Dashes in the germline sequence (CDR3) indicate that thesequences are not known or do not exist in the germline gene.

FIG. 5 shows deduced amino acid sequences of the TNV mAb light chainvariable regions. The amino acid sequences shown (single letterabbreviations) were deduced from DNA sequence determined from bothuncloned PCR products and cloned PCR products. The amino sequences areshown partitioned into the secretory signal sequence (signal), framework(FW), and complementarity determining region (CDR) domains. The aminoacid sequence for the Vg/38K-type light chain germline gene is shown onthe top line for each domain. Dots indicate that the amino acid in theTNV mAb is identical to the germline gene. TNV148(B) indicates that thesequence shown pertains to both TNV148 and TNV148B. ‘All’ indicates thatthe sequence shown pertains to TNV14, TNV15, TNV148, TNV148B, andTNV186.

FIG. 6 shows schematic illustrations of the heavy and light chainexpression plasmids used to make the rTNV148B-expressing C466 cells.p1783 is the heavy chain plasmid and p1776 is the light chain plasmid.The rTNV148B variable and constant region coding domains are shown asblack boxes. The immunoglobulin enhancers in the J-C introns are shownas gray boxes. Relevant restriction sites are shown. The plasmids areshown oriented such that transcription of the Ab genes proceeds in aclockwise direction. Plasmid p1783 is 19.53 kb in length and plasmidp1776 is 15.06 kb in length. The complete nucleotide sequences of bothplasmids are known. The variable region coding sequence in p1783 can beeasily replaced with another heavy chain variable region sequence byreplacing the BsiWI/BstBI restriction fragment. The variable regioncoding sequence in p1776 can be replaced with another variable regionsequence by replacing the SalI/AflII restriction fragment.

FIG. 7 shows graphical representation of growth curve analyses of fiverTNV148B-producing cell lines. Cultures were initiated on day 0 byseeding cells into T75 flasks in I5Q+MHX media to have a viable celldensity of 1.0×10⁵ cells/ml in a 30 ml volume. The cell cultures usedfor these studies had been in continuous culture since transfections andsubclonings were performed. On subsequent days, cells in the T flaskswere thoroughly resuspended and a 0.3 ml aliquot of the culture wasremoved. The growth curve studies were terminated when cell countsdropped below 1.5×10⁵ cells/ml. The number of live cells in the aliquotwas determined by typan blue exclusion and the remainder of the aliquotstored for later mAb concentration determination. An ELISA for human IgGwas performed on all sample aliquots at the same time.

FIG. 8 shows a graphical representation of the comparison of cell growthrates in the presence of varying concentrations of MHX selection. Cellsubclones C466A and C466B were thawed into MHX-free media (IMDM, 5% FBS,2 mM glutamine) and cultured for two additional days. Both cell cultureswere then divided into three cultures that contained either no MHX,0.2×MHX, or 1×MHX. One day later, fresh T75 flasks were seeded with thecultures at a starting density of 1×10⁵ cells/ml and cells counted at 24hour intervals for one week. Doubling times during the first 5 days werecalculated using the formula in SOP PD32.025 and are shown above thebars.

FIG. 9 shows graphical representations of the stability of mAbproduction over time from two rTNV148B-producing cell lines. Cellsubclones that had been in continuous culture since performingtransfections and subclonings were used to start long-term serialcultures in 24-well culture dishes. Cells were cultured in I5Q mediawith and without MHX selection. Cells were continually passaged bysplitting the cultures every 4 to 6 days to maintain new viable cultureswhile previous cultures were allowed to go spent. Aliquots of spent cellsupernatant were collected shortly after cultures were spent and storeduntil the mAb concentrations were determined. An ELISA for human IgG wasperformed on all sample aliquots at the same time.

FIG. 10 shows arthritis mouse model mice Tg197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 4. At approximately 4 weeks of age the Tg197 studymice were assigned, based on gender and body weight, to one of 9treatment groups and treated with a single intraperitoneal bolus dose ofDulbecco's PBS (D-PBS) or an anti-TNF antibody of the present invention(TNV14, TNV148 or TNV196) at either 1 mg/kg or 10 mg/kg. When theweights were analyzed as a change from pre-dose, the animals treatedwith 10 mg/kg cA2 showed consistently higher weight gain than theD-PBS-treated animals throughout the study. This weight gain wassignificant at weeks 3-7. The animals treated with 10 mg/kg TNV148 alsoachieved significant weight gain at week 7 of the study.

FIGS. 11A-C represent the progression of disease severity based on thearthritic index as presented in Example 4. The 10 mg/kg cA2-treatedgroup's arthritic index was lower than the D-PBS control group startingat week 3 and continuing throughout the remainder of the study (week 7).The animals treated with 1 mg/kg TNV14 and the animals treated with 1mg/kg cA2 failed to show significant reduction in AI after week 3 whencompared to the D-PBS-treated Group. There were no significantdifferences between the 10 mg/kg treatment groups when each was comparedto the others of similar dose (10 mg/kg cA2 compared to 10 mg/kg TNV14,148 and 196). When the 1 mg/kg treatment groups were compared, the 1mg/kg TNV148 showed a significantly lower AI than 1 mg/kg cA2 at 3, 4and 7 weeks. The 1 mg/kg TNV148 was also significantly lower than the 1mg/kg TNV14-treated Group at 3 and 4 weeks. Although TNV196 showedsignificant reduction in AI up to week 6 of the study (when compared tothe D-PBS-treated Group), TNV148 was the only 1 mg/kg treatment thatremained significant at the conclusion of the study.

FIG. 12 shows arthritis mouse model mice Tg197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 5. At approximately 4 weeks of age the Tg197 studymice were assigned, based on body weight, to one of 8 treatment groupsand treated with a intraperitoneal bolus dose of control article (D-PBS)or antibody (TNV14, TNV148) at 3 mg/kg (week 0). Injections wererepeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 wereevaluated for test article efficacy. Serum samples, obtained fromanimals in Groups 7 and 8 were evaluated for immune response inductionand pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4.

FIGS. 13A-C are graphs representing the progression of disease severityin Example 5 based on the arthritic index. The 10 mg/kg cA2-treatedgroup's arthritic index was significantly lower than the D-PBS controlgroup starting at week 2 and continuing throughout the remainder of thestudy (week 5). The animals treated with 1 mg/kg or 3 mg/kg of cA2 andthe animals treated with 3 mg/kg TNV14 failed to achieve any significantreduction in AI at any time throughout the study when compared to thed-PBS control group. The animals treated with 3 mg/kg TNV148 showed asignificant reduction when compared to the d-PBS-treated group startingat week 3 and continuing through week 5. The 10 mg/kg cA2-treatedanimals showed a significant reduction in AI when compared to both thelower doses (1 mg/kg and 3 mg/kg) of cA2 at weeks 4 and 5 of the studyand was also significantly lower than the TNV14-treated animals at weeks3-5. Although there appeared to be no significant differences betweenany of the 3 mg/kg treatment groups, the AI for the animals treated with3 mg/kg TNV14 were significantly higher at some time points than the 10mg/kg whereas the animals treated with TNV148 were not significantlydifferent from the animals treated with 10 mg/kg of cA2.

FIG. 14 shows arthritis mouse model mice Tg197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 6. At approximately 4 weeks of age the Tg197 studymice were assigned, based on gender and body weight, to one of 6treatment groups and treated with a single intraperitoneal bolus dose ofantibody (cA2, or TNV148) at either 3 mg/kg or 5 mg/kg. This studyutilized the D-PBS and 10 mg/kg cA2 control Groups.

FIG. 15 represents the progression of disease severity based on thearthritic index as presented in Example 6. All treatment groups showedsome protection at the earlier time points, with the 5 mg/kg cA2 and the5 mg/kg TNV148 showing significant reductions in AI at weeks 1-3 and alltreatment groups showing a significant reduction at week 2. Later in thestudy the animals treated with 5 mg/kg cA2 showed some protection, withsignificant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) ofboth the cA2 and the TNV148 showed significant reductions at 6 and alltreatment groups showed significant reductions at week 7. None of thetreatment groups were able to maintain a significant reduction at theconclusion of the study (week 8). There were no significant differencesbetween any of the treatment groups (excluding the saline control group)at any time point.

FIG. 16 shows arthritis mouse model mice Tg197 weight changes inresponse to anti-TNF antibodies of the present invention as compared tocontrols in Example 7. To compare the efficacy of a singleintraperitoneal dose of TNV148 (derived from hybridoma cells) andrTNV148B (derived from transfected cells). At approximately 4 weeks ofage the Tg197 study mice were assigned, based on gender and body weight,to one of 9 treatment groups and treated with a single intraperitonealbolus dose of Dulbecco's PBS (D-PBS) or antibody (TNV148, rTNV148B) at 1mg/kg.

FIG. 17 represents the progression of disease severity based on thearthritic index as presented in Example 7. The 10 mg/kg cA2-treatedgroup's arthritic index was lower than the D-PBS control group startingat week 4 and continuing throughout the remainder of the study (week 8).Both of the TNV148-treated Groups and the 1 mg/kg cA2-treated Groupshowed a significant reduction in AI at week 4. Although a previousstudy (P-099-017) showed that TNV148 was slightly more effective atreducing the Arthritic Index following a single 1 mg/kg intraperitonealbolus, this study showed that the AI from both versions of the TNVantibody-treated groups was slightly higher. Although (with theexception of week 6) the 1 mg/kg cA2-treated Group was not significantlyincreased when compared to the 10 mg/kg cA2 group and the TNV148-treatedGroups were significantly higher at weeks 7 and 8, there were nosignificant differences in AI between the 1 mg/kg cA2, 1 mg/kg TNV148and 1 mg/kg TNV148B at any point in the study.

FIG. 18 shows diagram of the study design for trial of Simponi(golimumab), administered intravenously, in subjects with activeAnkylosing Spondylitis (AS).

DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticanti-TNF human, primate, rodent, mammalian, chimeric, humanized orCDR-grafted, antibodies comprising all of the heavy chain variable CDRregions of SEQ ID NOS:1, 2 and 3 and/or all of the light chain variableCDR regions of SEQ ID NOS:4, 5 and 6 and TNF anti-idiotype antibodiesthereto, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding at least one anti-TNFantibody or anti-idiotype antibody. The present invention furtherincludes, but is not limited to, methods of making and using suchnucleic acids and antibodies and anti-idiotype antibodies, includingdiagnostic and therapeutic compositions, methods and devices.

As used herein, an “anti-tumor necrosis factor alpha antibody,”“anti-TNF antibody,” “anti-TNF antibody portion,” or “anti-TNF antibodyfragment” and/or “anti-TNF antibody variant” and the like include anyprotein or peptide containing molecule that comprises at least a portionof an immunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, or at least one portion of an TNFreceptor or binding protein, which can be incorporated into an antibodyof the present invention. Such antibody optionally further affects aspecific ligand, such as but not limited to where such antibodymodulates, decreases, increases, antagonizes, angonizes, mitigates,alleviates, blocks, inhibits, abrogates and/or interferes with at leastone TNF activity or binding, or with TNF receptor activity or binding,in vitro, in situ and/or in vivo. As a non-limiting example, a suitableanti-TNF antibody, specified portion or variant of the present inventioncan bind at least one TNF, or specified portions, variants or domainsthereof. A suitable anti-TNF antibody, specified portion, or variant canalso optionally affect at least one of TNF activity or function, such asbut not limited to, RNA, DNA or protein synthesis, TNF release, TNFreceptor signaling, membrane TNF cleavage, TNF activity, TNF productionand/or synthesis. The term “antibody “is further intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian TNF. For example, antibody fragmentscapable of binding to TNF or portions thereof, including, but notlimited to Fab (e.g., by papain digestion), Fab′ (e.g., by pepsindigestion and partial reduction) and F(ab′)2 (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) issubstantially non-immunogenic in humans, with only minor sequencechanges or variations. Similarly, antibodies designated primate (monkey,baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig,hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an F_(V) cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one TNF protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

1491 Anti-TNF antibodies (also termed TNF antibodies) useful in themethods and compositions of the present invention can optionally becharacterized by high affinity binding to TNF and optionally andpreferably having low toxicity. In particular, an antibody, specifiedfragment or variant of the invention, where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively, optionally and preferably possess lowimmunogenicity, is useful in the present invention. The antibodies thatcan be used in the invention are optionally characterized by theirability to treat patients for extended periods with measurablealleviation of symptoms and low and/or acceptable toxicity. Low oracceptable immunogenicity and/or high affinity, as well as othersuitable properties, can contribute to the therapeutic results achieved.“Low immunogenicity” is defined herein as raising significant HAHA, HACAor HAMA responses in less than about 75%, or preferably less than about50% of the patients treated and/or raising low titres in the patienttreated (less than about 300, preferably less than about 100 measuredwith a double antigen enzyme immunoassay) (Elliott et al., Lancet344:1125-1127 (1994), entirely incorporated herein by reference).

Utility: The isolated nucleic acids of the present invention can be usedfor production of at least one anti-TNF antibody or specified variantthereof, which can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one TNF condition, selected from, but not limitedto, at least one of an immune disorder or disease, a cardiovasculardisorder or disease, an infectious, malignant, and/or neurologicdisorder or disease.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-TNF antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple, or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts. Citations. All publications or patents cited herein areentirely incorporated herein by reference as they show the state of theart at the time of the present invention and/or to provide descriptionand enablement of the present invention. Publications refer to anyscientific or patent publications, or any other information available inany media format, including all recorded, electronic or printed formats.The following references are entirely incorporated herein by reference:Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlowand Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.(1989); Colligan, et al., eds., Current Protocols in Immunology, JohnWiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocolsin Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

Antibodies of the Present Invention: At least one anti-TNF antibody ofthe present invention comprising all of the heavy chain variable CDRregions of SEQ ID NOS:1, 2 and 3 and/or all of the light chain variableCDR regions of SEQ ID NOS:4, 5 and 6 can be optionally produced by acell line, a mixed cell line, an immortalized cell or clonal populationof immortalized cells, as well known in the art. See, e.g., Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001),each entirely incorporated herein by reference.

Human antibodies that are specific for human TNF proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas isolated and/or TNF protein or a portion thereof (including syntheticmolecules, such as synthetic peptides). Other specific or generalmammalian antibodies can be similarly raised. Preparation of immunogenicantigens, and monoclonal antibody production can be performed using anysuitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SAS, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260(5/12/94); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (Biolnvent); WO88/06630; W090/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and applications)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V.,Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,

-   -   www.ncbi.nlm.nih.gov/entrez/query.fcgi;        www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;        www.antibodyresource.com/onlinecomp.html;        www.public.iastate.edu/˜pedro/research_tools.html;        www.mgen.uni-heidelberg.de/SD/IT/IT.html;        www.whfreeman.com/immunology/CH05/kuby05.htm;        www.library.thinkquest.org/12429/Immune/Antibody.html;        www.hhmi.org/grants/lectures/1996/vlab/;        www.path.cam.ac.uk/˜mrc7/mikeimages.html;        www.antibodyresource.com/;        mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;        pathbox.wustl.edu/˜hcenter/index.html;        www.biotech.ufl.edu/˜hcl/; www.pebio.com/pa/340913/340913.html;        www.nal.usda.gov/awic/pubs/antibody/;        www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html;        www.biodesign.com/table.asp;        www.icnet.uk/axp/facs/davies/links.html;        www.biotech.ufl.edu/˜fccl/protocol.html;        www.isac-net.org/sites_geo.html;        aximtl.imt.uni-marburg.de/˜rek/AEPStart.html;        baserv.uci.kun.n1/˜jraats/links1.html;        www.recab.uni-hd.de/immuno.bme.nwu.edu/;        www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;        www.ibt.unam.mx/vir/V_mice.html; imgt. cnusc.fr:8104/;        www.biochem.ucl.ac.uk/˜martin/abs/index.html;        antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;        www.unizh.ch/˜honegger/AHOseminar/Slide01.html;        www.cryst.bbk.ac.uk/˜ubcg07s/;        www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;        www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;        www.ibt.unam.mx/vir/structure/stat_aim.html;        www.biosci.missouri.edu/smithgp/index.html;        www.cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;        www.jerini.de/fr_products.htm;        www.patents.ibm.com/ibm.html.Kabat et al.,

Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Humanization or engineering of antibodiesof the present invention can be performed using any known method, suchas but not limited to those described in, Winter (Jones et al., Nature321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al.,Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5714352, 6204023,6180370, 5693762, 5530101, 5585089, 5225539; 4816567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

The anti-TNF antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, hamster, non-human primate,and the like) capable of producing a repertoire of human antibodies, asdescribed herein and/or as known in the art. Cells that produce a humananti-TNF antibody can be isolated from such animals and immortalizedusing suitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 Bl,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474Bl, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol.6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456, assigned to Enzon; U.S. Pat. Nos. 5,223,409, 5,403,484,5,571,698, 5,837,500, assigned to Dyax, 5427908, 5580717, assigned toAffymax; 5885793, assigned to Cambridge antibody Technologies; 5750373,assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493,5698417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook,supra, each of the above patents and publications entirely incorporatedherein by reference.

Antibodies of the present invention can also be prepared using at leastone anti-TNF antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-TNF antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to know methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. See, also generally forplant expression of antibodies, but not limited to, Each of the abovereferences is entirely incorporated herein by reference.

The antibodies of the invention can bind human TNF with a wide range ofaffinities (K_(D)). In a preferred embodiment, at least one human mAb ofthe present invention can optionally bind human TNF with high affinity.For example, a human mAb can bind human TNF with a K_(D) equal to orless than about 10⁻⁷M, such as but not limited to, 0.1-9.9 (or any rangeor value therein)×10⁻⁷, 10⁻⁸, 10⁻⁹,10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or anyrange or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Nucleic Acid Molecules. Using the information provided herein, such asthe nucleotide sequences encoding at least 70-100% of the contiguousamino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8,specified fragments, variants or consensus sequences thereof, or adeposited vector comprising at least one of these sequences, a nucleicacid molecule of the present invention encoding at least one anti-TNFantibody comprising all of the heavy chain variable CDR regions of SEQID NOS:1, 2 and 3 and/or all of the light chain variable CDR regions ofSEQ ID NOS:4, 5 and 6 can be obtained using methods described herein oras known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-TNF antibody or variable region (e.g., SEQ ID NOS:7,8); andnucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-TNFantibody as described herein and/or as known in the art. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-TNF antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules of the present invention include SEQ ID NOS:10,11, 12, 13, 14, 15, corresponding to non-limiting examples of a nucleicacid encoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LCCDR2, LC CDR3, HC variable region and LC variable region.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-TNF antibody can include,but are not limited to, those encoding the amino acid sequence of anantibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides Which Selectively Hybridize to a Polynucleotide asDescribed Herein. The present invention provides isolated nucleic acidsthat hybridize under selective hybridization conditions to apolynucleotide disclosed herein. Thus, the polynucleotides of thisembodiment can be used for isolating, detecting, and/or quantifyingnucleic acids comprising such polynucleotides. For example,polynucleotides of the present invention can be used to identify,isolate, or amplify partial or full-length clones in a depositedlibrary. In some embodiments, the polynucleotides are genomic or cDNAsequences isolated, or otherwise complementary to, a cDNA from a humanor mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids. The isolated nucleic acids of the presentinvention can be made using (a) recombinant methods, (b) synthetictechniques, (c) purification techniques, or combinations thereof, aswell-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids. The isolated nucleicacid compositions of this invention, such as RNA, cDNA, genomic DNA, orany combination thereof, can be obtained from biological sources usingany number of cloning methodologies known to those of skill in the art.In some embodiments, oligonucleotide probes that selectively hybridize,under stringent conditions, to the polynucleotides of the presentinvention are used to identify the desired sequence in a cDNA or genomicDNA library. The isolation of RNA, and construction of cDNA and genomiclibraries, is well known to those of ordinary skill in the art. (See,e.g., Ausubel, supra; or Sambrook, supra).

Nucleic Acid Screening and Isolation Methods. A cDNA or genomic librarycan be screened using a probe based upon the sequence of apolynucleotide of the present invention, such as those disclosed herein.Probes can be used to hybridize with genomic DNA or cDNA sequences toisolate homologous genes in the same or different organisms. Those ofskill in the art will appreciate that various degrees of stringency ofhybridization can be employed in the assay; and either the hybridizationor the wash medium can be stringent. As the conditions for hybridizationbecome more stringent, there must be a greater degree of complementaritybetween the probe and the target for duplex formation to occur. Thedegree of stringency can be controlled by one or more of temperature,ionic strength, pH and the presence of a partially denaturing solventsuch as formamide. For example, the stringency of hybridization isconveniently varied by changing the polarity of the reactant solutionthrough, for example, manipulation of the concentration of formamidewithin the range of 0% to 50%. The degree of complementarity (sequenceidentity) required for detectable binding will vary in accordance withthe stringency of the hybridization medium and/or wash medium. Thedegree of complementarity will optimally be 100%, or 70-100%, or anyrange or value therein. However, it should be understood that minorsequence variations in the probes and primers can be compensated for byreducing the stringency of the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat.No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S.Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, etal; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids. The isolated nucleicacids of the present invention can also be prepared by direct chemicalsynthesis by known methods (see, e.g., Ausubel, et al., supra). Chemicalsynthesis generally produces a single-stranded oligonucleotide, whichcan be converted into double-stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill in the art will recognizethat while chemical synthesis of DNA can be limited to sequences ofabout 100 or more bases, longer sequences can be obtained by theligation of shorter sequences.

Recombinant Expression Cassettes. The present invention further providesrecombinant expression cassettes comprising a nucleic acid of thepresent invention. A nucleic acid sequence of the present invention, forexample a cDNA or a genomic sequence encoding an antibody of the presentinvention, can be used to construct a recombinant expression cassettethat can be introduced into at least one desired host cell. Arecombinant expression cassette will typically comprise a polynucleotideof the present invention operably linked to transcriptional initiationregulatory sequences that will direct the transcription of thepolynucleotide in the intended host cell. Both heterologous andnon-heterologous (i.e., endogenous) promoters can be employed to directexpression of the nucleic acids of the present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors And Host Cells. The present invention also relates to vectorsthat include isolated nucleic acid molecules of the present invention,host cells that are genetically engineered with the recombinant vectors,and the production of at least one anti-TNF antibody by recombinanttechniques, as is well known in the art. See, e.g., Sambrook, et al.,supra; Ausubel, et al., supra, each entirely incorporated herein byreference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating site at the beginning and a termination codon(e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNAto be translated, with UAA and UAG preferred for mammalian or eukaryoticcell expression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va (www.atcc.org). Preferred host cells include cells of lymphoid originsuch as myeloma and lymphoma cells. Particularly preferred host cellsare P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14cells (ATCC Accession Number CRL-1851). In a particularly preferredembodiment, the recombinant cell is a P3X63Ab8.653 or a SP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Antibody. An anti-TNF antibody can be recovered andpurified from recombinant cell cultures by well-known methods including,but not limited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Anti-TNF Antibodies

The isolated antibodies of the present invention, comprising all of theheavy chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all ofthe light chain variable CDR regions of SEQ ID NOS:4, 5 and 6, compriseantibody amino acid sequences disclosed herein encoded by any suitablepolynucleotide, or any isolated or prepared antibody. Preferably, thehuman antibody or antigen-binding fragment binds human TNF and, therebypartially or substantially neutralizes at least one biological activityof the protein. An antibody, or specified portion or variant thereof,that partially or preferably substantially neutralizes at least onebiological activity of at least one TNF protein or fragment can bind theprotein or fragment and thereby inhibit activities mediated through thebinding of TNF to the TNF receptor or through other TNF-dependent ormediated mechanisms. As used herein, the term “neutralizing antibody”refers to an antibody that can inhibit an TNF-dependent activity byabout 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or moredepending on the assay. The capacity of an anti-TNF antibody to inhibitan TNF-dependent activity is preferably assessed by at least onesuitable TNF protein or receptor assay, as described herein and/or asknown in the art. A human antibody of the invention can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. In one embodiment, the human antibody comprises anIgG heavy chain or defined fragment, for example, at least one ofisotypes, IgG1, IgG2, IgG3 or IgG4. Antibodies of this type can beprepared by employing a transgenic mouse or other transgenic non-humanmammal comprising at least one human light chain (e.g., IgG, IgA□0 andIgM (e.g., γ1, γ2, γ3, γ4) transgenes as described herein and/or asknown in the art. In another embodiment, the anti-human TNF humanantibody comprises an IgG1 heavy chain and a IgG1 light chain.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one TNF protein, subunit, fragment, portionor any combination thereof. The at least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofsaid protein, which epitope is preferably comprised of at least oneextracellular, soluble, hydrophilic, external or cytoplasmic portion ofsaid protein. The at least one specified epitope can comprise anycombination of at least one amino acid sequence of at least 1-3 aminoacids to the entire specified portion of contiguous amino acids of theSEQ ID NO:9.

Generally, the human antibody or antigen-binding fragment of the presentinvention will comprise an antigen-binding region that comprises atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one heavy chain variable region and at least onehuman complementarity determining region (CDR1, CDR2 and CDR3) orvariant of at least one light chain variable region. As a non-limitingexample, the antibody or antigen-binding portion or variant can compriseat least one of the heavy chain CDR3 having the amino acid sequence ofSEQ ID NO:3, and/or a light chain CDR3 having the amino acid sequence ofSEQ ID NO:6. In a particular embodiment, the antibody or antigen-bindingfragment can have an antigen-binding region that comprises at least aportion of at least one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NOS:1, 2, and/or 3). In another particular embodiment, theantibody or antigen-binding portion or variant can have anantigen-binding region that comprises at least a portion of at least onelight chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 4,5, and/or 6). In a preferred embodiment the three heavy chain CDRs andthe three light chain CDRs of the antibody or antigen-binding fragmenthave the amino acid sequence of the corresponding CDR of at least one ofmAb TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, TNV86, as describedherein. Such antibodies can be prepared by chemically joining togetherthe various portions (e.g., CDRs, framework) of the antibody usingconventional techniques, by preparing and expressing a (i.e., one ormore) nucleic acid molecule that encodes the antibody using conventionaltechniques of recombinant DNA technology or by using any other suitablemethod.

The anti-TNF antibody can comprise at least one of a heavy or lightchain variable region having a defined amino acid sequence. For example,in a preferred embodiment, the anti-TNF antibody comprises at least oneof heavy chain variable region, optionally having the amino acidsequence of SEQ ID NO:7 and/or at least one light chain variable region,optionally having the amino acid sequence of SEQ ID NO:8. antibodiesthat bind to human TNF and that comprise a defined heavy or light chainvariable region can be prepared using suitable methods, such as phagedisplay (Katsube, Y., et al., Int J Mal. Med, 1(5):863-868 (1998)) ormethods that employ transgenic animals, as known in the art and/or asdescribed herein. For example, a transgenic mouse, comprising afunctionally rearranged human immunoglobulin heavy chain transgene and atransgene comprising DNA from a human immunoglobulin light chain locusthat can undergo functional rearrangement, can be immunized with humanTNF or a fragment thereof to elicit the production of antibodies. Ifdesired, the antibody producing cells can be isolated and hybridomas orother immortalized antibody-producing cells can be prepared as describedherein and/or as known in the art. Alternatively, the antibody,specified portion or variant can be expressed using the encoding nucleicacid or portion thereof in a suitable host cell.

The invention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human TNF with high affinity(e.g., K_(D) less than or equal to about 10⁻⁹ M). Amino acid sequencesthat are substantially the same as the sequences described hereininclude sequences comprising conservative amino acid substitutions, aswell as amino acid deletions and/or insertions. A conservative aminoacid substitution refers to the replacement of a first amino acid by asecond amino acid that has chemical and/or physical properties (e.g.,charge, structure, polarity, hydrophobicity/hydrophilicity) that aresimilar to those of the first amino acid. Conservative substitutionsinclude replacement of one amino acid by another within the followinggroups: lysine (K), arginine (R) and histidine (H); aspartate (D) andglutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T),tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L),isoleucine (I), proline (P), phenylalanine (F), tryptophan (W),methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino Acid Codes. The amino acids that make up anti-TNF antibodies ofthe present invention are often abbreviated. The amino acid designationscan be indicated by designating the amino acid by its single lettercode, its three letter code, name, or three nucleotide codon(s) as iswell understood in the art (see Alberts, B., et al., Molecular Biologyof The Cell, Third Ed., Garland Publishing, Inc., New York, 1994):

SINGLE THREE LETTER LETTER THREE NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D AspAspartic acid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe PhenylalanineUUC, UUU G Gly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I IleIsoleucine AUA, AUC, AUU K Lys Lysine AAA, AAG L Leu LeucineUUA, UUG, CUA, CUC, CUG, CUU M Met Methionine AUG N Asn AsparagineAAC, AAU P Pro Proline CCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R ArgArginine AGA, AGG, CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA,UCC, UCG, UCU T Thr Threonine ACA, ACC, ACG, ACU V Val ValineGUA, GUC, GUG, GUU W Trp Tryptophan UGG Y Tyr Tyrosine UAC, UAU

An anti-TNF antibody of the present invention can include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-TNF antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-TNF antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one TNF neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

Anti-TNF antibodies of the present invention can include, but are notlimited to, at least one portion, sequence or combination selected from1 to all of the contiguous amino acids of at least one of SEQ ID NOS:1,2, 3, 4, 5, 6.

A(n) anti-TNF antibody can further optionally comprise a polypeptide ofat least one of 70-100% of the contiguous amino acids of at least one ofSEQ ID NOS:7, 8.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7, 8. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:7. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 7, 8. The antibodies of the present invention,or specified variants thereof, can comprise any number of contiguousamino acid residues from an antibody of the present invention, whereinthat number is selected from the group of integers consisting of from10-100% of the number of contiguous residues in an anti-TNF antibody.Optionally, this subsequence of contiguous amino acids is at least about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids inlength, or any range or value therein. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

Anti-Idiotype Antibodies To Anti-Tnf Antibody Compositions. In additionto monoclonal or chimeric anti-TNF antibodies, the present invention isalso directed to an anti-idiotypic (anti-Id) antibody specific for suchantibodies of the invention. An anti-Id antibody is an antibody whichrecognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal of the same species and genetic type (e.g. mousestrain) as the source of the Id antibody with the antibody or a CDRcontaining region thereof. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

Anti-Tnf Antibody Compositions. The present invention also provides atleast one anti-TNF antibody composition comprising at least one, atleast two, at least three, at least four, at least five, at least six ormore anti-TNF antibodies thereof, as described herein and/or as known inthe art that are provided in a non-naturally occurring composition,mixture or form. Such compositions comprise non-naturally occurringcompositions comprising at least one or two full length, C- and/orN-terminally deleted variants, domains, fragments, or specifiedvariants, of the anti-TNF antibody amino acid sequence selected from thegroup consisting of 70-100% of the contiguous amino acids of SEQ IDNOS:1, 2, 3, 4, 5, 6, 7, 8, or specified fragments, domains or variantsthereof. Preferred anti-TNF antibody compositions include at least oneor two full length, fragments, domains or variants as at least one CDRor LBR containing portions of the anti-TNF antibody sequence of 70-100%of SEQ ID NOS:1, 2, 3, 4, 5, 6, or specified fragments, domains orvariants thereof. Further preferred compositions comprise 40-99% of atleast one of 70-100% of SEQ ID NOS:1, 2, 3, 4, 5, 6, or specifiedfragments, domains or variants thereof. Such composition percentages areby weight, volume, concentration, molarity, or molality as liquid or drysolutions, mixtures, suspension, emulsions or colloids, as known in theart or as described herein.

Anti-TNF antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-TNF antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment or therapy, optionally further comprising atleast one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, anantiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide,a penicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropieitin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examplesof such cytokines include, but are not limited to, any of IL-1 to IL-23.Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium difficile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliocbacter species,(e.g., Heliocbacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersinia enterocolitica, Vibrio species (e.g., Vibrio cholerae, Vibrioparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, N.Y. (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-TNF antibody compounds, compositions or combinations of the presentinvention can further comprise at least one of any suitable auxiliary,such as, but not limited to, diluent, binder, stabilizer, buffers,salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(t1) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-TNF antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-TNF antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-TNF antibody compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-TNF antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents.

Formulations. As noted above, the invention provides for stableformulations, which is preferably a phosphate buffer with saline or achosen salt, as well as preserved solutions and formulations containinga preservative as well as multi-use preserved formulations suitable forpharmaceutical or veterinary use, comprising at least one anti-TNFantibody in a pharmaceutically acceptable formulation. Preservedformulations contain at least one known preservative or optionallyselected from the group consisting of at least one phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuricnitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride(e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and thelike), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof in an aqueousdiluent. Any suitable concentration or mixture can be used as known inthe art, such as 0.001-5%, or any range or value therein, such as, butnot limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09,0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5,4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examplesinclude, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5,0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0,2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol(e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s)(e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02,0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-TNF antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one anti-TNF antibody, and asecond vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one anti-TNF antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

The at least one anti-TNF antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one anti-TNF antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyols, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-TNF antibody and a preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-TNF antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-TNF antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one anti-TNFantibody that is reconstituted with a second vial containing water, apreservative and/or excipients, preferably a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

The solutions of at least one anti-TNF antibody in the invention can beprepared by a process that comprises mixing at least one antibody in anaqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-TNFantibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-TNF antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject® NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one anti-TNF antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

1401 The formulations of the present invention can be prepared by aprocess that comprises mixing at least one anti-TNF antibody and aselected buffer, preferably a phosphate buffer containing saline or achosen salt. Mixing the at least one antibody and buffer in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-TNF antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one anti-TNF antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications. The present invention also provides a methodfor modulating or treating at least one TNF related disease, in a cell,tissue, organ, animal, or patient, as known in the art or as describedherein, using at least one dual integrin antibody of the presentinvention.

The present invention also provides a method for modulating or treatingat least one TNF related disease, in a cell, tissue, organ, animal, orpatient including, but not limited to, at least one of obesity, animmune related disease, a cardiovascular disease, an infectious disease,a malignant disease or a neurologic disease.

The present invention also provides a method for modulating or treatingat least one immune related disease, in a cell, tissue, organ, animal,or patient including, but not limited to, at least one of rheumatoidarthritis, juvenile, systemic onset juvenile rheumatoid arthritis,Ankylosing Spondylitis, ankylosing spondilitis, gastric ulcer,seronegative arthropathies, osteoarthritis, inflammatory bowel disease,ulcerative colitis, systemic lupus erythematosis, antiphospholipidsyndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonaryfibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis,orchitis/vasectomy reversal procedures, allergic/atopic diseases,asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergicconjunctivitis, hypersensitivity pneumonitis, transplants, organtransplant rejection, graft-versus-host disease, systemic inflammatoryresponse syndrome, sepsis syndrome, gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiationexposure, acute pancreatitis, adult respiratory distress syndrome,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease,thrombocytopenia, graft rejection of any organ or tissue, kidneytransplant rejection, heart transplant rejection, liver transplantrejection, pancreas transplant rejection, lung transplant rejection,bone marrow transplant (BMT) rejection, skin allograft rejection,cartilage transplant rejection, bone graft rejection, small boweltransplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, primary biliary cirrhosis, thyroiditis,encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lungdisease, chronic obstructive pulmonary disease (COPD), familialhematophagocytic lymphohistiocytosis, dermatologic conditions,psoriasis, alopecia, nephrotic syndrome, nephritis, glomerularnephritis, acute renal failure, hemodialysis, uremia, toxicity,preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy,chemotherapy, radiation therapy (e.g., including but not limitedtoasthenia, anemia, cachexia, and the like), chronic salicylateintoxication, and the like. See, e.g., the Merck Manual, 12th-17thEditions, Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992,1999), Pharmacotherapy Handbook, Wells et al., eds., Second Edition,Appleton and Lange, Stamford, Conn. (1998, 2000), each entirelyincorporated by reference.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic ateriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specificarrhythmias, ventricular fibrillation, His bundle arrhythmias,atrioventricular block, bundle branch block, myocardial ischemicdisorders, coronary artery disease, angina pectoris, myocardialinfarction, cardiomyopathy, dilated congestive cardiomyopathy,restrictive cardiomyopathy, valvular heart diseases, endocarditis,pericardial disease, cardiac tumors, aortic and peripheral aneuryisms,aortic dissection, inflammation of the aorta, occlusion of the abdominalaorta and its branches, peripheral vascular disorders, occlusivearterial disorders, peripheral atherosclerotic disease, thromboangitisobliterans, functional peripheral arterial disorders, Raynaud'sphenomenon and disease, acrocyanosis, erythromelalgia, venous diseases,venous thrombosis, varicose veins, arteriovenous fistula, lymphedema,lipedema, unstable angina, reperfusion injury, post pump syndrome,ischemia-reperfusion injury, and the like. Such a method can optionallycomprise administering an effective amount of a composition orpharmaceutical composition comprising at least one anti-TNF antibody toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A,B or C, or the like),septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli 0157:h7,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shocksyndrome, streptococcal myositis, gas gangrene, Mycobacteriumtuberculosis, Mycobacterium avium intracellulare, Pneumocystis cariniipneumonia, pelvic inflammatory disease, orchitis/epidydimitis,legionella, lyme disease, influenza a, epstein-barr virus,viral-associated hemaphagocytic syndrome, vital encephalitis/asepticmeningitis, and the like.

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome(MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

The present invention also provides a method for modulating or treatingat least one neurologic disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasiaa, andmitochondrial multi-system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit' such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16^(th) Edition,Merck & Company, Rahway, N.J. (1992)

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-TNF antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases, wherein theadministering of said at least one anti-TNF antibody, specified portionor variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, antigen-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g.,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/orpreferably in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

Chimeric antibody cA2 consists of the antigen binding variable region ofthe high-affinity neutralizing mouse anti-human TNFα IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric antibody cA2 and recombinant human TNFα, the affinityconstant of chimeric antibody cA2 was calculated to be 1.04×10¹⁰M⁻¹.Preferred methods for determining monoclonal antibody specificity andaffinity by competitive inhibition can be found in Harlow, et al.,antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, NewYork, (1992-2000); Kozbor et al., Immunol. Today, 4:72-79 (1983);Ausubel et al., eds. Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2000); and Muller, Meth. Enzymol.,92:589-601 (1983), which references are entirely incorporated herein byreference.

In a particular embodiment, murine monoclonal antibody A2 is produced bya cell line designated c134A. Chimeric antibody cA2 is produced by acell line designated c168A.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

TNF Receptor Molecules. Preferred TNF receptor molecules useful in thepresent invention are those that bind TNFα with high affinity (see,e.g., Feldmann et al., International Publication No. WO 92/07076(published Apr. 30, 1992); Schall et al., Cell 61:361-370 (1990); andLoetscher et al., Cell 61:351-359 (1990), which references are entirelyincorporated herein by reference) and optionally possess lowimmunogenicity. In particular, the 55 kDa (p55 TNF-R) and the 75 kDa(p75 TNF-R) TNF cell surface receptors are useful in the presentinvention. Truncated forms of these receptors, comprising theextracellular domains (ECD) of the receptors or functional portionsthereof (see, e.g., Corcoran et al., Eur. J. Biochem. 223:831-840(1994)), are also useful in the present invention. Truncated forms ofthe TNF receptors, comprising the ECD, have been detected in urine andserum as 30 kDa and 40 kDa TNFα inhibitory binding proteins (Engelmann,H. et al., J. Biol. Chem. 265:1531-1536 (1990)). TNF receptor multimericmolecules and TNF immunoreceptor fusion molecules, and derivatives andfragments or portions thereof, are additional examples of TNF receptormolecules which are useful in the methods and compositions of thepresent invention. The TNF receptor molecules which can be used in theinvention are characterized by their ability to treat patients forextended periods with good to excellent alleviation of symptoms and lowtoxicity. Low immunogenicity and/or high affinity, as well as otherundefined properties, can contribute to the therapeutic resultsachieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. JImmunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. JImmunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNF□ with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000).

Cytokines include any known cytokine. See, e.g., CopewithCytokines.com.

Cytokine antagonists include, but are not limited to, any antibody,fragment or mimetic, any soluble receptor, fragment or mimetic, anysmall molecule antagonist, or any combination thereof.

Therapeutic Treatments. Any method of the present invention can comprisea method for treating a TNF mediated disorder, comprising administeringa safe and effective amount of a composition or pharmaceuticalcomposition comprising at least one anti-TNF antibody to a cell, tissue,organ, animal or patient in need of such modulation, treatment ortherapy. Such a method can optionally further comprise co-administrationor combination therapy for treating such immune diseases, wherein theadministering of said at least one anti-TNF antibody, specified portionor variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist.

As used herein, the term “safe”, as it relates to a composition, dose,dosage regimen, treatment or method with an anti-TNF antibody of thepresent invention (e.g., the anti-TNF antibody golimumab), refers to afavorable risk:benefit ratio with an acceptable frequency and/oracceptable severity of adverse events (AEs) and serious adverse events(SAEs) compared to the standard of care or to another comparator such asother anti-TNF agents. An adverse event is an untoward medicaloccurrence in a patient administered a medicinal product. In particular,safe as it relates to a composition, dose, dosage regimen, treatment ormethod with an anti-TNF antibody of the present invention refers to anacceptable frequency and/or acceptable severity of adverse eventsincluding, for example, infusion reactions, hepatobiliary laboratoryabnormalities, infections including TB, and malignancies.

The terms “efficacy” and “effective” as used herein in the context of acomposition, dose, dosage regimen, treatment or method refer to theeffectiveness of a particular composition, dose, dosage, treatment ormethod with an anti-TNF antibody of the present invention (e.g., theanti-TNF antibody golimumab). Efficacy can be measured based on changein the course of the disease in response to an agent of the presentinvention. For example, an anti-TNF antibody of the present invention isadministered to a patient in an amount and for a time sufficient toinduce an improvement, preferably a sustained improvement, in at leastone indicator that reflects the severity of the disorder that is beingtreated. Various indicators that reflect the extent of the subject'sillness, disease or condition may be assessed for determining whetherthe amount and time of the treatment is sufficient. Such indicatorsinclude, for example, clinically recognized indicators of diseaseseverity, symptoms, or manifestations of the disorder in question. Thedegree of improvement generally is determined by a physician or otheradequately trained individual, who may make the determination based onsigns, symptoms, biopsies, or other test results that indicateamelioration of clinical symptoms or any other measure of diseaseactivity. For example, an anti-TNF antibody of the present invention maybe administered to achieve an improvement in a patient's conditionrelated to Ankylosing Spondylitis (AS). Improvement in a patient'scondition related to AS can be assessed using one or more criteriaincluding, for example, an Ankylosing Spondylitis Disease Activity Score(ASDAS), a Bath Ankylosing Spondylitis Functional Index (BASFI), a BathAnkylosing Spondylitis Metrology Index (BASMI), a 36-item Short-FormHealth Survey Physical Component Summary (SF-36 PCS), a 36-itemShort-Form Health Survey Mental Component Summary (SF-36 MCS), and/orresults from an Ankylosing Spondylitis Qualify of Life (ASQoL)questionnaire. ASDAS is a disease activity score (DAS) for use in ASthat was developed by the Assessment of SpondyloArthritis internationalSociety. ASDAS is calculated using a formula with assessments thatinclude, e.g., total back pain, duration of morning stiffness,peripheral pain/swelling and a patient global assessment. BASFI is asubject's self-assessment represented as a mean of 10 questions, 8 ofwhich relate to the subject's functional anatomy and 2 of which relateto a subject's ability to cope with everyday life. BASMI is an aggregatescore calculated by converting assessments into scores for 5 assessmentsincluding, lateral lumbar flexion, tragus-to-wall distance, lumbarflexion, intermalleolar distance, and cervical rotation angle. SF-36 isa questionnaire consisting of 8 multi-item scales that are scored andSF-36 PSA and SF-36 MCS are summary scores derived from the SF-36 thatallow comparisons of the relative burden of different diseases and therelative benefit of different treatments. ASQoL is a self-administeredpatient-reported outcomes instrument consisting of 18 items requesting aresponse to questions related to the impact of pain on sleep, mood,motivation, ability to cope, activities of daily living, independence,relationships, and social life.

Typically, treatment of pathologic conditions is effected byadministering a safe and effective amount or dosage of at least oneanti-TNF antibody composition that total, on average, a range from atleast about 0.01 to 500 milligrams of at least one anti-TNF antibody perkilogram of patient per dose, and preferably from at least about 0.1 to100 milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 μg/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 15, 15.5,15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9,20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000μg/ml serum concentration per single or multiple administration, or anyrange, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration. Many known and developed modes ofadministration can be used according to the present invention foradministering pharmaceutically effective amounts of at least oneanti-TNF antibody according to the present invention. While pulmonaryadministration is used in the following description, other modes ofadministration can be used according to the present invention withsuitable results.

TNF antibodies of the present invention can be delivered in a carrier,as a solution, emulsion, colloid, or suspension, or as a dry powder,using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration. Formulations for parenteraladministration can contain as common excipients sterile water or saline,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes and the like. Aqueous or oilysuspensions for injection can be prepared by using an appropriateemulsifier or humidifier and a suspending agent, according to knownmethods. Agents for injection can be a non-toxic, non-orallyadministrable diluting agent such as aqueous solution or a sterileinjectable solution or suspension in a solvent. As the usable vehicle orsolvent, water, Ringer's solution, isotonic saline, etc. are allowed; asan ordinary solvent, or suspending solvent, sterile involatile oil canbe used. For these purposes, any kind of involatile oil and fatty acidcan be used, including natural or synthetic or semisynthetic fatty oilsor fatty acids; natural or synthetic or semisynthtetic mono- or di- ortri-glycerides. Parental administration is known in the art andincludes, but is not limited to, conventional means of injections, a gaspressured needle-less injection device as described in U.S. Pat. No.5,851,198, and a laser perforator device as described in U.S. Pat. No.5,839,446 entirely incorporated herein by reference.

Alternative Delivery. The invention further relates to theadministration of at least one anti-TNF antibody by parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermalmeans. At least one anti-TNF antibody composition can be prepared foruse for parenteral (subcutaneous, intramuscular or intravenous) or anyother administration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

1761 Pulmonary/Nasal Administration. For pulmonary administration,preferably at least one anti-TNF antibody composition is delivered in aparticle size effective for reaching the lower airways of the lung orsinuses. According to the invention, at least one anti-TNF antibody canbe delivered by any of a variety of inhalation or nasal devices known inthe art for administration of a therapeutic agent by inhalation. Thesedevices capable of depositing aerosolized formulations in the sinuscavity or alveoli of a patient include metered dose inhalers,nebulizers, dry powder generators, sprayers, and the like. Other devicessuitable for directing the pulmonary or nasal administration ofantibodies are also known in the art. All such devices can use offormulations suitable for the administration for the dispensing ofantibody in an aerosol. Such aerosols can be comprised of eithersolutions (both aqueous and non-aqueous) or solid particles. Metereddose inhalers like the Ventolin® metered dose inhaler, typically use apropellant gas and require actuation during inspiration (See, e.g., WO94/16970, WO 98/35888). Dry powder inhalers like Turbuhaler (Astra),Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devicesmarketed by Inhale Therapeutics, and the Spinhaler® powder inhaler(Fisons), use breath-actuation of a mixed powder (U.S. Pat. No.4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura,U.S. Pat. No. 5,458,135 Inhale, WO 94/06498 Fisons, entirelyincorporated herein by reference). Nebulizers like AERx™ Aradigm, theUltravent® nebulizer (Mallinckrodt), and the Acorn II® nebulizer(Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO97/22376), the above references entirely incorporated herein byreference, produce aerosols from solutions, while metered dose inhalers,dry powder inhalers, etc. generate small particle aerosols. Thesespecific examples of commercially available inhalation devices areintended to be a representative of specific devices suitable for thepractice of this invention, and are not intended as limiting the scopeof the invention. Preferably, a composition comprising at least oneanti-TNF antibody is delivered by a dry powder inhaler or a sprayer.There are a several desirable features of an inhalation device foradministering at least one antibody of the present invention. Forexample, delivery by the inhalation device is advantageously reliable,reproducible, and accurate. The inhalation device can optionally deliversmall dry particles, e.g. less than about 10 μm, preferably about 1-5μm, for good respirability.

Administration of TNF antibody Compositions as a Spray. A sprayincluding TNF antibody composition protein can be produced by forcing asuspension or solution of at least one anti-TNF antibody through anozzle under pressure. The nozzle size and configuration, the appliedpressure, and the liquid feed rate can be chosen to achieve the desiredoutput and particle size. An electrospray can be produced, for example,by an electric field in connection with a capillary or nozzle feed.Advantageously, particles of at least one anti-TNF antibody compositionprotein delivered by a sprayer have a particle size less than about 10μm, preferably in the range of about 1 μm to about 5 μm, and mostpreferably about 2 μm to about 3 μm.

Formulations of at least one anti-TNF antibody composition proteinsuitable for use with a sprayer typically include antibody compositionprotein in an aqueous solution at a concentration of about 0.1 mg toabout 100 mg of at least one anti-TNF antibody composition protein perml of solution or mg/gm, or any range or value therein, e.g., but notlimited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml ormg/gm. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the antibody composition protein, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating antibody composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulatingantibody composition proteins include sucrose, mannitol, lactose,trehalose, glucose, or the like. The antibody composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the antibody composition protein causedby atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as TNF antibodies, or specified portionsor variants, can also be included in the formulation.

Administration of TNF antibody compositions by a Nebulizer. Antibodycomposition protein can be administered by a nebulizer, such as jetnebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, acompressed air source is used to create a high-velocity air jet throughan orifice. As the gas expands beyond the nozzle, a low-pressure regionis created, which draws a solution of antibody composition proteinthrough a capillary tube connected to a liquid reservoir. The liquidstream from the capillary tube is sheared into unstable filaments anddroplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 μm, preferably in therange of about 1 to about 5 μm, and most preferably about 2 μm to about3 μm.

Formulations of at least one anti-TNF antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one anti-TNF antibodyprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-TNFantibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-TNF antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneanti-TNF antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one anti-TNF antibody formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the at least one anti-TNF antibody caused by atomizationof the solution in forming an aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbital fatty acid esters. Amounts will generallyrange between 0.001 and 4% by weight of the formulation. Especiallypreferred surfactants for purposes of this invention are polyoxyethylenesorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.Additional agents known in the art for formulation of a protein such asantibody protein can also be included in the formulation.

Administration of TNF antibody compositions By A Metered Dose Inhaler.

In a metered dose inhaler (MDI), a propellant, at least one anti-TNFantibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one anti-TNF antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-TNF antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-TNF antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some casessolution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one anti-TNF antibody compositions via devices not describedherein.

Oral Formulations and Administration. Formulations for oral rely on theco-administration of adjuvants (e.g., resorcinols and nonionicsurfactants such as polyoxyethylene oleyl ether andn-hexadecylpolyethylene ether) to increase artificially the permeabilityof the intestinal walls, as well as the co-administration of enzymaticinhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 are used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration. For absorption through mucosalsurfaces, compositions and methods of administering at least oneanti-TNF antibody include an emulsion comprising a plurality ofsubmicron particles, a mucoadhesive macromolecule, a bioactive peptide,and an aqueous continuous phase, which promotes absorption throughmucosal surfaces by achieving mucoadhesion of the emulsion particles(U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application ofthe emulsions of the present invention can include corneal,conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic,intestinal, and rectal routes of administration. Formulations forvaginal or rectal administration, e.g. suppositories, can contain asexcipients, for example, polyalkyleneglycols, vaseline, cocoa butter,and the like. Formulations for intranasal administration can be solidand contain as excipients, for example, lactose or can be aqueous oroily solutions of nasal drops. For buccal administration excipientsinclude sugars, calcium stearate, magnesium stearate, pregelinatinedstarch, and the like (U.S. Pat. No. 5,849,695).

Transdermal Formulations and Administration. For transdermaladministration, the at least one anti-TNF antibody is encapsulated in adelivery device such as a liposome or polymeric nanoparticles,microparticle, microcapsule, or microspheres (referred to collectivelyas microparticles unless otherwise stated). A number of suitable devicesare known, including microparticles made of synthetic polymers such aspolyhydroxy acids such as polylactic acid, polyglycolic acid andcopolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. No. 5,814,599).

Prolonged Administration and Formulations. It can be sometimes desirableto deliver the compounds of the present invention to the subject overprolonged periods of time, for example, for periods of one week to oneyear from a single administration. Various slow release, depot orimplant dosage forms can be utilized. For example, a dosage form cancontain a pharmaceutically acceptable non-toxic salt of the compoundsthat has a low degree of solubility in body fluids, for example, (a) anacid addition salt with a polybasic acid such as phosphoric acid,sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids,polygalacturonic acid, and the like; (b) a salt with a polyvalent metalcation such as zinc, calcium, bismuth, barium, magnesium, aluminum,copper, cobalt, nickel, cadmium and the like, or with an organic cationformed from e.g., N,N′-dibenzyl-ethylenediamine or ethylenediamine; or(c) combinations of (a) and

(b) e.g. a zinc tannate salt. Additionally, the compounds of the presentinvention or, preferably, a relatively insoluble salt such as those justdescribed, can be formulated in a gel, for example, an aluminummonostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1: Cloning and Expression of TNF Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES lneo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells. The vector pC4 is used for theexpression of TNF antibody. Plasmid pC4 is a derivative of the plasmidpSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouseDHFR gene under control of the SV40 early promoter. Chinese hamsterovary- or other cells lacking dihydrofolate activity that aretransfected with these plasmids can be selected by growing the cells ina selective medium (e.g., alpha minus MEM, Life Technologies,Gaithersburg, Md.) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., F. W. Alt, etal., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma,Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A.Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, Xbal, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human beta-actin promoter,the SV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the TNF ina regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 2: Generation of High Affinity Human IgG Monoclonal AntibodiesReactive With Human TNF Using Transgenic Mice

Summary Transgenic mice have been used that contain human heavy andlight chain immunoglobulin genes to generate high affinity, completelyhuman, monoclonal antibodies that can be used therapeutically to inhibitthe action of TNF for the treatment of one or more TNF-mediated disease.(CBA/J×C57/BL6/J) F₂ hybrid mice containing human variable and constantregion antibody transgenes for both heavy and light chains are immunizedwith human recombinant TNF (Taylor et al., Intl. Immunol. 6:579-591(1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M.,Nature Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology14:845-851 (1996)). Several fusions yielded one or more panels ofcompletely human TNF reactive IgG monoclonal antibodies. The completelyhuman anti-TNF antibodies are further characterized. All are IgG1κ. Suchantibodies are found to have affinity constants somewhere between 1×10⁹and 9×10¹². The unexpectedly high affinities of these fully humanmonoclonal antibodies make them suitable candidates for therapeuticapplications in TNF related diseases, pathologies or disorders.

Abbreviations. BSA—bovine serum albumin; CO₂—carbon dioxide;DMSO—dimethyl sulfoxide; EIA—enzyme immunoassay; FBS—fetal bovine serum;H₂O₂—hydrogen peroxide; HRP—horseradish peroxidase; ID—interadermal;Ig—immunoglobulin; TNF—tissue necrosis factor alpha; IP—intraperitoneal;IV—intravenous; Mab—monoclonal antibody; OD—optical density;OPD—o-Phenylenediamine dihydrochloride; PEG—polyethylene glycol;PSA—penicillin, streptomycin, amphotericin; RT—room temperature;SQ—subcutaneous; v/v—volume per volume; w/v—weight per volume.

Materials and Methods

Animals. Transgenic mice that can express human antibodies are known inthe art (and are commercially available (e.g., from GenPharmInternational, San Jose, Calif.; Abgenix, Freemont, Calif., and others)that express human immunoglobulins but not mouse IgM or Igκ. Forexample, such transgenic mice contain human sequence transgenes thatundergo V(D)J joining, heavy-chain class switching, and somatic mutationto generate a repertoire of human sequence immunoglobulins (Lonberg, etal., Nature 368:856-859 (1994)). The light chain transgene can bederived, e.g., in part from a yeast artificial chromosome clone thatincludes nearly half of the germline human Vκ region. In addition, theheavy-chain transgene can encode both human μ and human γ1(Fishwild, etal., Nature Biotechnology 14:845-851 (1996)) and/or γ3 constant regions.Mice derived from appropriate genotopic lineages can be used in theimmunization and fusion processes to generate fully human monoclonalantibodies to TNF.

Immunization. One or more immunization schedules can be used to generatethe anti-TNF human hybridomas. The first several fusions can beperformed after the following exemplary immunization protocol, but othersimilar known protocols can be used. Several 14-20 week old femaleand/or surgically castrated transgenic male mice are immunized IP and/orID with 1-1000 μg of recombinant human TNF emulsified with an equalvolume of TITERMAX or complete Freund's adjuvant in a final volume of100-400 μL (e.g., 200). Each mouse can also optionally receive 1-10 μgin 100 μL physiological saline at each of 2 SQ sites. The mice can thenbe immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400μg) and SQ (1-400 μg×2) with TNF emulsified with an equal volume ofTITERMAX or incomplete Freund's adjuvant. Mice can be bled 12-25 and25-40 days later by retro-orbital puncture without anti-coagulant. Theblood is then allowed to clot at RT for one hour and the serum iscollected and titered using an TNF EIA assay according to known methods.Fusions are performed when repeated injections do not cause titers toincrease. At that time, the mice can be given a final IV boosterinjection of 1-400 μg TNF diluted in 100 μL physiological saline. Threedays later, the mice can be euthanized by cervical dislocation and thespleens removed aseptically and immersed in 10 mL of cold phosphatebuffered saline (PBS) containing 100 U/mL penicillin, 100 μg/mLstreptomycin, and 0.25 μg/mL amphotericin B (PSA). The splenocytes areharvested by sterilely perfusing the spleen with PSA-PBS. The cells arewashed once in cold PSA-PBS, counted using Trypan blue dye exclusion andresuspended in RPMI 1640 media containing 25 mM Hepes.

Cell Fusion. Fusion can be carried out at a 1:1 to 1:10 ratio of murinemyeloma cells to viable spleen cells according to known methods, e.g.,as known in the art. As a non-limiting example, spleen cells and myelomacells can be pelleted together. The pellet can then be slowlyresuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEGmolecular weight 1,450, Sigma) at 37° C. The fusion can then be stoppedby slowly adding 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37°C.) over 1 minute. The fused cells are centrifuged for 5 minutes at500-1500 rpm. The cells are then resuspended in HAT medium (RPMI 1640medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mMsodium pyruvate, 4 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origenculturing supplement (Fisher), 10% 653-conditioned RPMI 1640/Hepesmedia, 50 μM 2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin,and 16 μM thymidine) and then plated at 200μL/well in fifteen 96-wellflat bottom tissue culture plates. The plates are then placed in ahumidified 37° C. incubator containing 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-TNF Antibodies in Mouse Serum. Solid phaseEIA's can be used to screen mouse sera for human IgG antibodies specificfor human TNF. Briefly, plates can be coated with TNF at 2 μg/mL in PBSovernight. After washing in 0.15M saline containing 0.02% (v/v) Tween20, the wells can be blocked with 1% (w/v) BSA in PBS, 200 4/well for 1hour at RT. Plates are used immediately or frozen at −20° C. for futureuse. Mouse serum dilutions are incubated on the TNF coated plates at 504/well at RT for 1 hour. The plates are washed and then probed with 504/well HRP-labeled goat anti-human IgG, Fc specific diluted 1:30,000 in1% BSA-PBS for 1 hour at RT. The plates can again be washed and 1004/well of the citrate-phosphate substrate solution (0.1M citric acid and0.2M sodium phosphate, 0.01% H₂O₂ and 1 mg/mL OPD) is added for 15minutes at RT. Stop solution (4N sulfuric acid) is then added at 25μL/well and the OD's are read at 490 nm via an automated platespectrophotometer.

Detection of Completely Human Immunoglobulins in Hybridoma Supernates.Growth positive hybridomas secreting fully human immunoglobulins can bedetected using a suitable EIA. Briefly, 96 well pop-out plates (VWR,610744) can be coated with 10 μg/mL goat anti-human IgG Fc in sodiumcarbonate buffer overnight at 4° C. The plates are washed and blockedwith 1% BSA-PBS for one hour at 37° C. and used immediately or frozen at−20° C. Undiluted hybridoma supernatants are incubated on the plates forone hour at 37° C. The plates are washed and probed with HRP labeledgoat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for one hour at 37°C. The plates are then incubated with substrate solution as describedabove.

Determination of Fully Human Anti-TNF Reactivity. Hybridomas, as above,can be simultaneously assayed for reactivity to TNF using a suitable RIAor other assay. For example, supernatants are incubated on goatanti-human IgG Fc plates as above, washed and then probed withradiolabled TNF with appropriate counts per well for 1 hour at RT. Thewells are washed twice with PBS and bound radiolabled TNF is quantitatedusing a suitable counter.

Human IgGκ anti-TNF secreting hybridomas can be expanded in cell cultureand serially subcloned by limiting dilution. The resulting clonalpopulations can be expanded and cryopreserved in freezing medium (95%FBS, 5% DMSO) and stored in liquid nitrogen.

Isotyping. Isotype determination of the antibodies can be accomplishedusing an EIA in a format similar to that used to screen the mouse immunesera for specific titers. TNF can be coated on 96-well plates asdescribed above and purified antibody at 2 μg/mL can be incubated on theplate for one hour at RT. The plate is washed and probed with HRPlabeled goat anti-human IgG₁ or HRP labeled goat anti-human IgG₃ dilutedat 1:4000 in 1% BSA-PBS for one hour at RT. The plate is again washedand incubated with substrate solution as described above.

Binding Kinetics of Human Anti-Human TNF Antibodies With Human TNF.Binding characteristics for antibodies can be suitably assessed using anTNF capture EIA and BIAcore technology, for example. Gradedconcentrations of purified human TNF antibodies can be assessed forbinding to EIA plates coated with 2 μg/mL of TNF in assays as describedabove. The OD's can be then presented as semi-log plots showing relativebinding efficiencies.

Quantitative binding constants can be obtained, e.g., as follows, or byany other known suitable method. A BIAcore CM-5 (carboxymethyl) chip isplaced in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cellof the chip at 5 μL/minute until a stable baseline is obtained. Asolution (100 μL) of 15 mg of EDC(N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200μL water is added to 100 μL of a solution of 2.3 mg of NHS(N-hydroxysuccinimide) in 200 μL water. Forty (40) μL of the resultingsolution is injected onto the chip. Six μL of a solution of human TNF(15 μg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip,resulting in an increase of ca. 500 RU. The buffer is changed toTBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mMcalcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 μg/mLBSA, pH 7.4) and flowed over the chip overnight to equilibrate it and tohydrolyze or cap any unreacted succinimide esters.

Antibodies are dissolved in the running buffer at 33.33, 16.67, 8.33,and 4.17 nM. The flow rate is adjusted to 30 μL/min and the instrumenttemperature to 25° C. Two flow cells are used for the kinetic runs, oneon which TNF had been immobilized (sample) and a second, underivatizedflow cell (blank). 120 μL of each antibody concentration is injectedover the flow cells at 30 4/min (association phase) followed by anuninterrupted 360 seconds of buffer flow (dissociation phase). Thesurface of the chip is regenerated (tissue necrosis factoralpha/antibody complex dissociated) by two sequential injections of 30μL each of 2 M guanidine thiocyanate.

Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, asknown in the art. For each antibody concentration the blank sensogram issubtracted from the sample sensogram. A global fit is done for bothdissociation (k_(d), sec⁻¹) and association (k_(a), mol⁻¹ sec⁻¹) and thedissociation constant (K_(D), mol) calculated (k_(d)/k_(a)). Where theantibody affinity is high enough that the RUs of antibody capturedare >100, additional dilutions of the antibody are run.

Results and Discussion

Generation of Anti-Human TNF Monoclonal Antibodies. Several fusions areperformed and each fusion is seeded in 15 plates (1440 wells/fusion)that yield several dozen antibodies specific for human TNF. Of these,some are found to consist of a combination of human and mouse Ig chains.The remaining hybridomas secret anti-TNF antibodies consisting solely ofhuman heavy and light chains. Of the human hybridomas all are expectedto be IgG1κ.

Binding Kinetics of Human Anti-Human TNF Antibodies. ELISA analysisconfirms that purified antibody from most or all of these hybridomasbind TNF in a concentration-dependent manner. FIGS. 1-2 show the resultsof the relative binding efficiency of these antibodies. In this case,the avidity of the antibody for its cognate antigen (epitope) ismeasured. It should be noted that binding TNF directly to the EIA platecan cause denaturation of the protein and the apparent bindingaffinities cannot be reflective of binding to undenatured protein. Fiftypercent binding is found over a range of concentrations.

Quantitative binding constants are obtained using BIAcore analysis ofthe human antibodies and reveals that several of the human monoclonalantibodies are very high affinity with K_(D) in the range of 1×1⁻⁹ to7×10⁻¹².

Conclusions.

Several fusions are performed utilizing splenocytes from hybrid micecontaining human variable and constant region antibody transgenes thatare immunized with human TNF. A set of several completely human TNFreactive IgG monoclonal antibodies of the IgG1κ isotype are generated.The completely human anti-TNF antibodies are further characterized.Several of generated antibodies have affinity constants between 1×10⁹and 9×10¹². The unexpectedly high affinities of these fully humanmonoclonal antibodies make them suitable for therapeutic applications inTNF-dependent diseases, pathologies or related conditions.

Example 3: Generation of Human IgG Monoclonal Antibodies Reactive toHuman TNFα

Summary (CBA/J×C57BL/6J) F₂ hybrid mice (1-4) containing human variableand constant region antibody transgenes for both heavy and light chainswere immunized with recombinant human TNFα. One fusion, named GenTNV,yielded eight totally human IgG1κ monoclonal antibodies that bind toimmobilized recombinant human TNFα. Shortly after identification, theeight cell lines were transferred to Molecular Biology for furthercharacterization. As these Mabs are totally human in sequence, they areexpected to be less immunogenic than cA2 (Remicade) in humans.

Abbreviations. BSA—bovine serum albumin; CO₂—carbon dioxide;DMSO—dimethyl sulfoxide; EIA—enzyme immunoassay; FBS—fetal bovine serum;H₂O₂-hydrogen peroxide; HC—heavy chain; HRP—horseradish peroxidase;ID—interadermal; Ig—immunoglobulin; TNF—tissue necrosis factor alpha;IP—intraperitoneal; IV—intravenous; Mab—monoclonal antibody; OD—opticaldensity; OPD—o-Phenylenediamine dihydrochloride; PEG—polyethyleneglycol; PSA—penicillin, streptomycin, amphotericin; RT—room temperature;SQ—subcutaneous; TNFα—tumor necrosis factor alpha; v/v—volume pervolume; w/v—weight per volume.

Introduction. Transgenic mice that contain human heavy and light chainimmunoglobulin genes were utilized to generate totally human monoclonalantibodies that are specific to recombinant human TNFα. It is hoped thatthese unique antibodies can be used, as cA2 (Remicade) is used totherapeutically inhibit the inflammatory processes involved inTNFα-mediated disease with the benefit of increased serum half-life anddecreased side effects relating to immunogenicity.

Materials and Methods.

Animals. Transgenic mice that express human immunoglobulins, but notmouse IgM or Igκ, have been developed by GenPharm International. Thesemice contain functional human antibody transgenes that undergo V(D)Jjoining, heavy-chain class switching and somatic mutation to generate arepertoire of antigen-specific human immunoglobulins (1). The lightchain transgenes are derived in part from a yeast artificial chromosomeclone that includes nearly half of the germline human Vx locus. Inaddition to several VH genes, the heavy-chain (HC) transgene encodesboth human μ and human γ1 (2) and/or γ3 constant regions. A mousederived from the HCo12/KCo5 genotypic lineage was used in theimmunization and fusion process to generate the monoclonal antibodiesdescribed here.

Purification of Human TNFα. Human TNFα was purified from tissue culturesupernatant from C237A cells by affinity chromatography using a columnpacked with the TNFα receptor-Fc fusion protein (p55-sf2) (5) coupled toSepharose 4B (Pharmacia). The cell supernatant was mixed with one-ninthits volume of 10×Dulbecco's PBS (D-PBS) and passed through the column at4° C. at 4 mL/min. The column was then washed with PBS and the TNFα waseluted with 0.1 M sodium citrate, pH 3.5 and neutralized with 2 MTris-HCl pH 8.5. The purified TNFα was buffer exchanged into 10 mM Tris,0.12 M sodium chloride pH 7.5 and filtered through a 0.2 um syringefilter.

Immunizations. A female GenPharm mouse, approximately 16 weeks old, wasimmunized IP (200 μL) and ID (100 μL at the base of the tail) with atotal of 100 μg of TNFα (lot JG102298 or JG102098) emulsified with anequal volume of Titermax adjuvant on days 0, 12 and 28. The mouse wasbled on days 21 and 35 by retro-orbital puncture without anti-coagulant.The blood was allowed to clot at RT for one hour and the serum wascollected and titered using TNFα solid phase EIA assay. The fusion,named GenTNV, was performed after the mouse was allowed to rest forseven weeks following injection on day 28. The mouse, with a specifichuman IgG titer of 1:160 against TNFα, was then given a final IV boosterinjection of 50 μg TNFα diluted in 100 μL physiological saline. Threedays later, the mouse was euthanized by cervical dislocation and thespleen was removed aseptically and immersed in 10 mL of coldphosphate-buffered saline (PBS) containing 100 U/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL amphotericin B (PSA). The splenocyteswere harvested by sterilely perfusing the spleen with PSA-PBS. The cellswere washed once in cold PSA-PBS, counted using a Coulter counter andresuspended in RPMI 1640 media containing 25 mM Hepes.

Cell Lines. The non-secreting mouse myeloma fusion partner, 653 wasreceived into Cell Biology Services (CBS) group on May 14, 1997 fromCentocor's Product Development group. The cell line was expanded in RPMImedium (JRH Biosciences) supplemented with 10% (v/v) FBS (Cell CultureLabs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine (all from JRHBiosciences) and cryopreserved in 95% FBS and 5% DMSO (Sigma), thenstored in a vapor phase liquid nitrogen freezer in CBS. The cell bankwas sterile (Quality Control Centocor, Malvern) and free of mycoplasma(Bionique Laboratories). Cells were maintained in log phase cultureuntil fusion. They were washed in PBS, counted, and viability determined(>95%) via trypan blue dye exclusion prior to fusion.

Human TNFα was produced by a recombinant cell line, named C237A,generated in Molecular Biology at Centocor. The cell line was expandedin IMDM medium (JRH Biosciences) supplemented with 5% (v/v) FBS (CellCulture Labs), 2 mM L-glutamine (all from JRH Biosciences), and 0.5:g/mLmycophenolic acid, and cryopreserved in 95% FBS and 5% DMSO (Sigma),then stored in a vapor phase liquid nitrogen freezer in CBS (13). Thecell bank was sterile (Quality Control Centocor, Malvern) and free ofmycoplasma (Bionique Laboratories).

Cell Fusion. The cell fusion was carried out using a 1:1 ratio of 653murine myeloma cells and viable murine spleen cells. Briefly, spleencells and myeloma cells were pelleted together. The pellet was slowlyresuspended over a 30 second period in 1 mL of 50% (w/v) PEG/PBSsolution (PEG molecular weight of 1,450 g/mole, Sigma) at 37° C. Thefusion was stopped by slowly adding 10.5 mL of RPMI media (no additives)(JRH) (37° C.) over 1 minute. The fused cells were centrifuged for 5minutes at 750 rpm. The cells were then resuspended in HAT medium(RPMI/HEPES medium containing 10% Fetal Bovine Serum (JRH), 1 mM sodiumpyruvate, 2 mM L-glutamine, 10 μg/mL gentamicin, 2.5% Origen culturingsupplement (Fisher), 50 μM 2-mercaptoethanol, 1% 653-conditioned RPMImedia, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) andthen plated at 200 μL/well in five 96-well flat bottom tissue cultureplates. The plates were then placed in a humidified 37° C. incubatorcontaining 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-TNFα Antibodies in Mouse Serum. Solid phaseEIAs were used to screen mouse sera for human IgG antibodies specificfor human TNFα. Briefly, plates were coated with TNFα at 1 μg/mL in PBSovernight. After washing in 0.15 M saline containing 0.02% (v/v) Tween20, the wells were blocked with 1% (w/v) BSA in PBS, 200 μL/well for 1hour at RT. Plates were either used immediately or frozen at −20° C. forfuture use. Mouse sera were incubated in two-fold serial dilutions onthe human TNFα-coated plates at 50 μL/well at RT for 1 hour. The plateswere washed and then probed with 50 μL/well HRP-labeled goat anti-humanIgG, Fc specific (Accurate) diluted 1:30,000 in 1% BSA-PBS for 1 hour atRT. The plates were again washed and 100 μL/well of thecitrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodiumphosphate, 0.01% H₂O₂ and 1 mg/mL OPD) was added for 15 minutes at RT.Stop solution (4N sulfuric acid) was then added at 25 μL/well and theOD's were read at 490 nm using an automated plate spectrophotometer.

Detection of Totally Human Immunoglobulins in Hybridoma Supernatants.Because the GenPharm mouse is capable of generating both mouse and humanimmunoglobulin chains, two separate EIA assays were used to testgrowth-positive hybridoma clones for the presence of both human lightchains and human heavy chains. Plates were coated as described above andundiluted hybridoma supernatants were incubated on the plates for onehour at 37° C. The plates were washed and probed with eitherHRP-conjugated goat anti-human kappa (Southern Biotech) antibody diluted1:10,000 in 1% BSA-HBSS or HRP-conjugated goat anti-human IgG Fcspecific antibody diluted to 1:30,000 in 1% BSA-HBSS for one hour at 37°C. The plates were then incubated with substrate solution as describedabove. Hybridoma clones that did not give a positive signal in both theanti-human kappa and anti-human IgG Fc EIA formats were discarded.

Isotyping. Isotype determination of the antibodies was accomplishedusing an EIA in a format similar to that used to screen the mouse immunesera for specific titers. EIA plates were coated with goat anti-humanIgG (H+L) at 10:g/mL in sodium carbonate buffer overnight at 4EC andblocked as described above. Neat supernatants from 24 well cultures wereincubated on the plate for one hour at RT. The plate was washed andprobed with HRP-labeled goat anti-human IgG₁, IgG₂, IgG₃ or IgG₄(Binding Site) diluted at 1:4000 in 1% BSA-PBS for one hour at RT. Theplate was again washed and incubated with substrate solution asdescribed above.

Results and Discussion. Generation of Totally Human Anti-Human TNFαMonoclonal Antibodies. One fusion, named GenTNV, was performed from aGenPharm mouse immunized with recombinant human TNFα protein. From thisfusion, 196 growth-positive hybrids were screened. Eight hybridoma celllines were identified that secreted totally human IgG antibodiesreactive with human TNFα. These eight cell lines each secretedimmunoglobulins of the human IgG1κ isotype and all were subcloned twiceby limiting dilution to obtain stable cell lines (>90% homogeneous).Cell line names and respective C code designations are listed inTable 1. Each of the cell lines was frozen in 12-vial research cellbanks stored in liquid nitrogen.

Parental cells collected from wells of a 24-well culture dish for eachof the eight cell lines were handed over to Molecular Biology group onFeb. 18, 1999 for transfection and further characterization.

TABLE 1 GenTNV Cell Line Designations Name C Code DesignationGenTNV14.17.12 C414A GenTNV15.28.11 C415A GenTNV32.2.16 C416AGenTNV86.14.34 C417A GenTNV118.3.36 C418A GenTNV122.23.2 C419AGenTNV148.26.12 C420A GenTNV196.9.1 C421A

Conclusion.

The GenTNV fusion was performed utilizing splenocytes from a hybridmouse containing human variable and constant region antibody transgenesthat was immunized with recombinant human TNFα prepared at Centocor.Eight totally human, TNFα-reactive IgG monoclonal antibodies of theIgG1κ isotype were generated. Parental cell lines were transferred toMolecular Biology group for further characterization and development.One of these new human antibodies may prove useful in anti-inflammatorywith the potential benefit of decreased immunogenicity and allergic-typecomplications as compared with Remicade.

REFERENCES

Taylor, et al., International Immunology 6:579-591 (1993).

Lonberg, et al., Nature 368:856-859 (1994).

Neuberger, M. Nature Biotechnology 14:826 (1996).

Fishwild, et al., Nature Biotechnology 14:845-851 (1996).

Scallon, et al., Cytokine 7:759-770 (1995).

Example 4: Cloning and Preparation of Cell Lines Expressing HumanAnti-TNFα Antibody

Summary A panel of eight human monoclonal antibodies (mAbs) with a TNVdesignation were found to bind immobilized human TNFα with apparentlyhigh avidity. Seven of the eight mAbs were shown to efficiently blockhuTNFα binding to a recombinant TNF receptor. Sequence analysis of theDNA encoding the seven mAbs confirmed that all the mAbs had human Vregions. The DNA sequences also revealed that three pairs of the mAbswere identical to each other, such that the original panel of eight mAbscontained only four distinct mAbs, represented by TNV14, TNV15, TNV148,and TNV196. Based on analyses of the deduced amino acid sequences of themAbs and results of in vitro TNFα neutralization data, mAb TNV148 andTNV14 were selected for further study.

Because the proline residue at position 75 (framework 3) in the TNV148heavy chain was not found at that position in other human antibodies ofthe same subgroup during a database search, site-directed DNAmutagenesis was performed to encode a serine residue at that position inorder to have it conform to known germline framework e sequences. Theserine modified mAb was designated TNV148B. PCR-amplified DNA encodingthe heavy and light chain variable regions of TNV148B and TNV14 wascloned into newly prepared expression vectors that were based on therecently cloned heavy and light chain genes of another human mAb(12B75), disclosed in U.S. patent application No. 60/236,827, filed Oct.7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses,published as WO 02/12500 which is entirely incorporated herein byreference.

P3X63Ag8.653 (653) cells or Sp2/0-Ag14 (Sp2/0) mouse myeloma cells weretransfected with the respective heavy and light chain expressionplasmids and screened through two rounds of subcloning for cell linesproducing high levels of recombinant TNV148B and TNV14 (rTNV148B andrTNV14) mAbs. Evaluations of growth curves and stability of mAbproduction over time indicated that 653-transfectant clones C466D andC466C stably produced approximately 125:g/ml of rTNV148B mAb in spentcultures whereas Sp2/0 transfectant 1.73-12-122 (C467A) stably producedapproximately 25:g/ml of rTNV148B mAb in spent cultures. Similaranalyses indicated that Sp2/0-transfectant clone C476A produced 18:g/mlof rTNV14 in spent cultures.

Introduction. A panel of eight mAbs derived from human TNFα-immunizedGenPharm/Medarex mice (HCo12/KCo5 genotype) were previously shown tobind human TNFα and to have a totally human IgG1, kappa isotype. Asimple binding assay was used to determine whether the exemplary mAbs ofthe invention were likely to have TNFα-neutralizing activity byevaluating their ability to block TNFα from binding to recombinant TNFreceptor. Based on those results, DNA sequence results, and in vitrocharacterizations of several of the mAbs, TNV148 was selected as the mAbto be further characterized.

DNA sequences encoding the TNV148 mAb were cloned, modified to fit intogene expression vectors that encode suitable constant regions,introduced into the well-characterized 653 and Sp2/0 mouse myelomacells, and resulting transfected cell lines screened until subcloneswere identified that produced 40-fold more mAb than the originalhybridoma cell line.

Materials and Methods.

Reagents and Cells. TRIZOL reagent was purchased from Gibco BRL.Proteinase K was obtained from Sigma Chemical Company. ReverseTranscriptase was obtained from Life Sciences, Inc. Taq DNA Polymerasewas obtained from either Perkin Elmer Cetus or Gibco BRL. Restrictionenzymes were purchased from New England Biolabs. QlAquick PCRPurification Kit was from Qiagen. A QuikChange Site-Directed MutagenesisKit was purchased from Stratagene. Wizard plasmid miniprep kits andRNasin were from Promega. Optiplates were obtained from Packard.¹²⁵Iodine was purchased from Amersham. Custom oligonucleotides werepurchased from Keystone/Biosource International. The names,identification numbers, and sequences of the oligonucleotides used inthis work are shown in Table 2.

TABLE 2Oligonucleotides used to clone, engineer, or sequence the TNV mAb genes.Name I.D. Sequence HG1-4b 119 3′-TTGGTCCAGTCGGACTGG-5′ (SEQ ID NO: 10)HG1-5b 354 3′-CACCTGCACTCGGTGCTT-5′ (SEQ ID NO: 11) HG1hg 3603′-CACTGTTTTGAGTGTGTACGGGCTTAAGTT-5′ (SEQ ID NO: 12) HG1-6  353′-GCCGCACGTGTGGAAGGG-5′ (SEQ ID NO: 13) HCK1-3E 1173′-AGTCAAGGTCGGACTGGCTTAAGTT-5′ (SEQ ID NO: 14) HuK-3′Hd 2083′-GTTGTCCCCTCTCACAATCTTCGAATTT-5′ (SEQ ID NO: 15) HVKRNAseq  343′-GGCGGTAGACTACTCGTC-5′ (SEQ ID NO: 16) BsiWI M D W T W S I(SEQ ID NO: 17) 5′14s 366 5-TTTCGTACGCCACCATGGACTGGACCTGGAGCATC-3′(SEQ ID NO: 18) 5′46s 367 5′-TTTCGTACGCCACCATGGGGTTTGGGCTGAGCTG-3′(SEQ ID NO: 19) 5′47s 368 5′-TTTCGTACGCCACCATGGAGTTTGGGCTGAGCATG-3′(SEQ ID NO: 20) 5′63s 369 5′-TTTCGTACGCCACCATGAAACACCTGTGGTTCTTC-3′(SEQ ID NO: 21) 5′73s 370 5′-TTTCGTACGCCACCATGGGGTCAACCGCCATCCTC-3′(SEQ ID NO: 22) T V T V S S BstBI (SEQ ID NO: 23) HuH-J6 3883′GTGCCAGTGGCAGAGGAGTCCATTCAAGCTTAAGTT-5′ (SEQ ID NO: 24) SalI M D M R V (SEQ ID NO: 25) LK7s 3625′-TTTGTCGACACCATGGACATGAGGGTCC(TC)C-3′ (SEQ ID NO: 26) LVgs 3635′-TTTGTCGACACCATGGAAGCCCCAGCTC-3′ (SEQ ID NO: 27)T K V D I K (SEQ ID NO: 28) Afl2 HuL-J3 3803′CTGGTTTCACCTATAGTTTG/CATTCAGAATTCGGCGCCTTT (SEQ ID NO: 29) V148-QC1399 5′-CATCTCCAGAGACAATtCCAAGAACACGCTGTATC-3′ (SEQ ID NO: 30) V148-QC2400 3′-GTAGAGGTCTCTGTTAaGGTTCTTGTGCGACATAG-5′ (SEQ ID NO: 31) The aminoacids encoded by oligonucleotide 5′14s and HuH-J6 are shown above thesequence. The ‘M’ amino acid residue represents the translation startcodon. The underlined sequences in oligonucleotides 5′14s and HuH-J6mark the BsiWI and BstBI restriction sites, respectively. The slash inHuH-J6 corresponds to the exon/intron boundary. Note thatoligonucleotides whose sequence corresponds to the minus strand arewritten in a 3′-5′ orientation.

A single frozen vial of 653 mouse myeloma cells was obtained. The vialwas thawed that day and expanded in T flasks in IMDM, 5% FBS, 2 mMglutamine (media). These cells were maintained in continuous cultureuntil they were transfected 2 to 3 weeks later with the anti-TNF DNAdescribed here. Some of the cultures were harvested 5 days after thethaw date, pelleted by centrifugation, and resuspended in 95% FBS, 5%DMSO, aliquoted into 30 vials, frozen, and stored for future use.Similarly, a single frozen vial of Sp2/0 mouse myeloma cells wasobtained. The vial was thawed, a new freeze-down prepared as describedabove, and the frozen vials stored in CBC freezer boxes AA and AB. Thesecells were thawed and used for all Sp2/0 transfections described here.

Assay for Inhibition of TNF Binding to Receptor. Hybridoma cellsupernatants containing the TNV mAbs were used to assay for the abilityof the mAbs to block binding of ¹²⁵I-labeled TNFα to the recombinant TNFreceptor fusion protein, p55-sf2 (Scallon et al. (1995) Cytokine7:759-770). 50:1 of p55-sf2 at 0.5:g/ml in PBS was added to Optiplatesto coat the wells during a one-hour incubation at 37° C. Serialdilutions of the eight TNV cell supernatants were prepared in 96-wellround-bottom plates using PBS/0.1% BSA as diluent. Cell supernatantcontaining anti-IL-18 mAb was included as a negative control and thesame anti-IL-18 supernatant spiked with cA2 (anti-TNF chimeric antibody,Remicade, U.S. Pat. No. 5,770,198, entirely incorporated herein byreference) was included as a positive control. ¹²⁵I-labeled TNFα(58:Ci/:g, D. Shealy) was added to 100:1 of cell supernatants to have afinal TNFα concentration of 5 ng/ml. The mixture was preincubated forone hour at RT. The coated Optiplates were washed to remove unboundp55-sf2 and 50:1 of the¹²⁵I-TNFα/cell supernatant mixture wastransferred to the Optiplates. After 2 hrs at RT, Optiplates were washedthree times with PBS-Tween. 100:1 of Microscint-20 was added and the cpmbound determined using the TopCount gamma counter.

Amplification of V Genes and DNA Sequence Analysis. Hybridoma cells werewashed once in PBS before addition of TRIZOL reagent for RNApreparation. Between 7×10⁶ and 1.7×10⁷ cells were resuspended in 1 mlTRIZOL. Tubes were shaken vigorously after addition of 200 μl ofchloroform. Samples were centrifuged at 4° C. for 10 minutes. Theaqueous phase was transferred to a fresh microfuge tube and an equalvolume of isopropanol was added. Tubes were shaken vigorously andallowed to incubate at room temperature for 10 minutes. Samples werethen centrifuged at 4° C. for 10 minutes. The pellets were washed oncewith 1 ml of 70% ethanol and dried briefly in a vacuum dryer. The RNApellets were resuspended with 40 μl of DEPC-treated water. The qualityof the RNA preparations was determined by fractionating 0.5 μl in a 1%agarose gel. The RNA was stored in a −80° C. freezer until used.

To prepare heavy and light chain cDNAs, mixtures were prepared thatincluded 3 μl of RNA and 1 μg of either oligonucleotide 119 (heavychain) or oligonucleotide 117 (light chain) (see Table 1) in a volume of11.5 μl. The mixture was incubated at 70° C. for 10 minutes in a waterbath and then chilled on ice for 10 minutes. A separate mixture wasprepared that was made up of 2.5 μl of 10× reverse transcriptase buffer,10 μl of 2.5 mM dNTPs, 1 μl of reverse transcriptase (20 units), and0.41 μl of ribonuclease inhibitor RNasin (1 unit). 13.5 μl of thismixture was added to the 11.5 μl of the chilled RNA/oligonucleotidemixture and the reaction incubated for 40 minutes at 42° C. The cDNAsynthesis reaction was then stored in a −20° C. freezer until used.

The unpurified heavy and light chain cDNAs were used as templates toPCR-amplify the variable region coding sequences. Five oligonucleotidepairs (366/354, 367/354, 368/354, 369/354, and 370/354, Table 1) weresimultaneously tested for their ability to prime amplification of theheavy chain DNA. Two oligonucleotide pairs (362/208 and 363/208) weresimultaneously tested for their ability to prime amplification of thelight chain DNA. PCR reactions were carried out using 2 units ofPLATINUM™ high fidelity (HIFI) Taq DNA polymerase in a total volume of50 μl. Each reaction included 2 μl of a cDNA reaction, 10 pmoles of eacholigonucleotide, 0.2 mM dNTPs, 5 μl of 10×HIFI Buffer, and 2 mMmagnesium sulfate. The thermal cycler program was 95° C. for 5 minutesfollowed by 30 cycles of (94° C. for 30 seconds, 62° C. for 30 seconds,68° C. for 1.5 minutes). There was then a final incubation at 68° C. for10 minutes.

To prepare the PCR products for direct DNA sequencing, they werepurified using the QIAquick™ PCR Purification Kit according to themanufacturer's protocol. The DNA was eluted from the spin column using50 μl of sterile water and then dried down to a volume of 10 μl using avacuum dryer. DNA sequencing reactions were then set up with 1 μl ofpurified PCR product, 10 μM oligonucleotide primer, 4 μl BigDyeTerminator™ ready reaction mix, and 14 μl sterile water for a totalvolume of 20 μl. Heavy chain PCR products made with oligonucleotide pair367/354 were sequenced with oligonucleotide primers 159 and 360. Lightchain PCR products made with oligonucleotide pair 363/208 were sequencedwith oligonucleotides 34 and 163. The thermal cycler program forsequencing was 25 cycles of (96° C. for 30 seconds, 50° C. for 15seconds, 60° C. for 4 minutes) followed by overnight at 4° C. Thereaction products were fractionated through a polyacrylamide gel anddetected using an ABI 377 DNA Sequencer.

Site-directed Mutagenesis to Change an Amino Acid. A single nucleotidein the TNV148 heavy chain variable region DNA sequence was changed inorder to replace Pro⁷⁵ with a Serine residue in the TNV148 mAb.Complimentary oligonucleotides, 399 and 400 (Table 1), were designed andordered to make this change using the QuikChange™ site-directedmutagenesis method as described by the manufacturer. The twooligonucleotides were first fractionated through a 15% polyacrylamidegel and the major bands purified. Mutagenesis reactions were preparedusing either 10 ng or 50 ng of TNV148 heavy chain plasmid template(p1753), 5 μl of 10× reaction buffer, 1 μl of dNTP mix, 125 ng of primer399, 125 ng of primer 400, and 1 μl of Pfu DNA Polymerase. Sterile waterwas added to bring the total volume to 50 μl. The reaction mix was thenincubated in a thermal cycler programmed to incubate at 95° C. for 30seconds, and then cycle 14 times with sequential incubations of 95° C.for 30 seconds, 55° C. for 1 minute, 64° C. for 1 minute, and 68° C. for7 minutes, followed by 30° C. for 2 minutes (1 cycle). These reactionswere designed to incorporate the mutagenic oligonucleotides intootherwise identical, newly synthesized plasmids. To rid of the originalTNV148 plasmids, samples were incubated at 37° C. for 1 hour afteraddition of 1 μl of Dpnl endonuclease, which cleaves only the originalmethylated plasmid. One μ.1 of the reaction was then used to transformEpicurian Coli XL1-Blue supercompetent E. coli by standard heat-shockmethods and transformed bacteria identified after plating onLB-ampicillin agar plates. Plasmid minipreps were prepared using theWizard™ kits as described by the manufacturer. After elution of samplefrom the Wizard™ column, plasmid DNA was precipitated with ethanol tofurther purify the plasmid DNA and then resuspended in 20 μl of sterilewater. DNA sequence analysis was then performed to identify plasmidclones that had the desired base change and to confirm that no otherbase changes were inadvertently introduced into the TNV148 codingsequence. One μl of plasmid was subjected to a cycle sequencing reactionprepared with 3 μl of BigDye mix, 1 μl of pUC19 Forward primer, and 10μl of sterile water using the same parameters described in Section 4.3.

Construction of Expression Vectors from 12B75 Genes. Several recombinantDNA steps were performed to prepare a new human IgG1 expression vectorand a new human kappa expression vector from the previously-clonedgenomic copies of the 12B75-encoding heavy and light chain genes,respectively, disclosed in U.S. patent application No. 60/236,827, filedOct. 7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses,published as WO 02/12500, which is entirely incorporated herein byreference. The final vectors were designed to permit simple, one-stepreplacement of the existing variable region sequences with anyappropriately-designed, PCR-amplified, variable region.

To modify the 12B75 heavy chain gene in plasmid p1560, a 6.85 kbBamHI/HindIII fragment containing the promoter and variable region wastransferred from p1560 to pUC19 to make p1743. The smaller size of thisplasmid compared to p1560 enabled use of QuikChange™ mutagenesis (usingoligonucleotides BsiWI-1 and BsiWI-2) to introduce a unique BsiWIcloning site just upstream of the translation initiation site, followingthe manufacturer's protocol. The resulting plasmid was termed p1747. Tointroduce a BstBI site at the 3′ end of the variable region, a 5′oligonucleotide primer was designed with SalI and BstB1 sites. Thisprimer was used with the pUC reverse primer to amplify a 2.75 kbfragment from p1747. This fragment was then cloned back into thenaturally-occurring SalI site in the 12B75 variable region and a HindIIIsite, thereby introducing the unique BstB1 site. The resultingintermediate vector, designated p1750, could accept variable regionfragments with BsiWI and BstBI ends. To prepare a version of heavy chainvector in which the constant region also derived from the 12B75 gene,the BamHI-HindIII insert in p1750 was transferred to pBR322 in order tohave an EcoRI site downstream of the HindIII site. The resultingplasmid, p1768, was then digested with HindIII and EcoRI and ligated toa 5.7 kb HindIII-EcoRI fragment from p1744, a subclone derived bycloning the large BamHI-BamHI fragment from p1560 into pBC. Theresulting plasmid, p1784, was then used as vector for the TNV Ab cDNAfragments with BsiWI and BstBI ends. Additional work was done to prepareexpression vectors, p1788 and p1798, which include the IgG1 constantregion from the 12B75 gene and differ from each other by how much of the12B75 heavy chain J-C intron they contain.

To modify the 12B75 light chain gene in plasmid p1558, a 5.7 kbSalI/AflII fragment containing the 12B75 promoter and variable regionwas transferred from p1558 into the XhoI/AflII sites of plasmid L28.This new plasmid, p1745, provided a smaller template for the mutagenesisstep. Oligonucleotides (C340salI and C340sal2) were used to introduce aunique SalI restriction site at the 5′ end of the variable region byQuikChange™ mutagenesis. The resulting intermediate vector, p1746, hadunique SalI and AflII restriction sites into which variable regionfragments could be cloned. Any variable region fragment cloned intop1746 would preferably be joined with the 3′ half of the light chaingene. To prepare a restriction fragment from the 3′ half of the 12B75light chain gene that could be used for this purpose, oligonucleotidesBAHN-1 and BAHN-2 were annealed to each other to form a double-strandedlinker containing the restriction sites BsiW1, AflII, HindII, and NotIand which contained ends that could be ligated into KpnI and SacI sites.This linker was cloned between the KpnI and SacI sites of pBC to giveplasmid p1757. A 7.1 kb fragment containing the 12B75 light chainconstant region, generated by digesting p1558 with AflII, then partiallydigesting with HindIII, was cloned between the AflII and HindII sites ofp1757 to yield p1762. This new plasmid contained unique sites for BsiWIand AflII into which the BsiWI/AflII fragment containing the promoterand variable regions could be transferred uniting the two halves of thegene.

cDNA Cloning and Assembly of Expression Plasmids. All RT-PCR reactions(see above) were treated with Klenow enzyme to further fill in the DNAends. Heavy chain PCR fragments were digested with restriction enzymesBsiWI and BstBI and then cloned between the BsiWI and BstBI sites ofplasmid L28 (L28 used because the 12B75-based intermediate vector p1750had not been prepared yet). DNA sequence analysis of the cloned insertsshowed that the resulting constructs were correct and that there were noerrors introduced during PCR amplifications. The assigned identificationnumbers for these L28 plasmid constructs (for TNV14, TNV15, TNV148,TNV148B, and TNV196) are shown in Table 3.

The BsiWI/BstBI inserts for TNV14, TNV148, and TNV148B heavy chains weretransferred from the L28 vector to the newly prepared intermediatevector, p1750. The assigned identification numbers for theseintermediate plasmids are shown in Table 2. This cloning step andsubsequent steps were not done for TNV15 and TNV196. The variableregions were then transferred into two different human IgG1 expressionvectors. Restriction enzymes EcoRI and HindIII were used to transfer thevariable regions into Centocor's previously-used IgG1 vector, p104. Theresulting expression plasmids, which encode an IgG1 of the Gm(f+)allotype, were designated p1781 (TNV14), p1782 (TNV148), and p1783(TNV148B) (see Table 2). The variable regions were also cloned upstreamof the IgG1 constant region derived from the 12B75 (GenPharm) gene.Those expression plasmids, which encode an IgG1 of the Glm(z) allotype,are also listed in Table 3.

TABLE 3 Plasmid identification numbers for various heavy and light chainplasmids. The L28 vector or pBC vector represents the initial Ab cDNAclone. The inserts in those plasmids were transferred to an incomplete12B75-based vector to make the intermediate plasmids. One additionaltransfer step resulted in the final expression plasmids that were eitherintroduced into cells after being linearized or used to purify the mAbgene inserts prior to cell transfection. (ND) = not done. Gm(f+) G1m(z)128 vector Intermediate Expression Expression Mab Plasmid ID Plasmid IDPlasmid ID Plasmid ID Heavy Chains TNV14 p1751 p1777 p1781 p1786 TNV15p1752 (ND) (ND) (ND) TNV148 p1753 p1778 p1782 p1787 TNV148B p1760 p1779p1783 p1788 TNV196 p1754 (ND) (ND) (ND) pBC vector IntermediateExpression Plasmid ID Plasmid ID Plasmid ID Light Chains TNV14 p1748p1755 p1775 TNV15 p1748 p1755 p1775 TNV148 p1749 p1756 p1776 TNV196p1749 p1756 p1776

Light chain PCR products were digested with restriction enzymes SalI andSacII and then cloned between the SalI and SacII sites of plasmid pBC.The two different light chain versions, which differed by one aminoacid, were designated p1748 and p1749 (Table 2). DNA sequence analysisconfirmed that these constructs had the correct sequences. TheSalI/AflII fragments in p1748 and p1749 were then cloned between theSalI and AflII sites of intermediate vector p1746 to make p1755 andp1756, respectively. These 5′ halves of the light chain genes were thenjoined to the 3′ halves of the gene by transferring the BsiWI/AflIIfragments from p1755 and p1756 to the newly prepared construct p1762 tomake the final expression plasmids p1775 and p1776, respectively (Table2).

Cell Transfections, Screening, and Subcloning. A total of 15transfections of mouse myeloma cells were performed with the various TNVexpression plasmids (see Table 3 in the Results and Discussion section).These transfections were distinguished by whether (1) the host cellswere Sp2/0 or 653; (2) the heavy chain constant region was encoded byCentocor's previous IgG1 vector or the 12B75 heavy chain constantregion; (3) the mAb was TNV148B, TNV148, TNV14, or a new HC/LCcombination; (4) whether the DNA was linearized plasmid or purified Abgene insert; and (5) the presence or absence of the complete J-C intronsequence in the heavy chain gene. In addition, several of thetransfections were repeated to increase the likelihood that a largenumber of clones could be screened.

Sp2/0 cells and 653 cells were each transfected with a mixture of heavyand light chain DNA (8-12:g each) by electroporation under standardconditions as previously described (Knight DM et al. (1993)MolecularImmunology 30:1443-1453). For transfection numbers 1, 2, 3, and 16, theappropriate expression plasmids were linearized by digestion with arestriction enzyme prior to transfection. For example, SalI and NotIrestriction enzymes were used to linearize TNV148B heavy chain plasmidp1783 and light chain plasmid p1776, respectively. For the remainingtransfections, DNA inserts that contained only the mAb gene wereseparated from the plasmid vector by digesting heavy chain plasmids withBamHI and light chain plasmids with BsiWI and NotI. The mAb gene insertswere then purified by agarose gel electrophoresis and Qiex purificationresins. Cells transfected with purified gene inserts were simultaneouslytransfected with 3-5:g of Psti-linearized pSV2gpt plasmid (p13) as asource of selectable marker. Following electroporation, cells wereseeded in 96-well tissue culture dishes in IMDM, 15% FBS, 2 mM glutamineand incubated at 37° C. in a 5% CO₂ incubator. Two days later, an equalvolume of IMDM, 5% FBS, 2 mM glutamine, 2×MHX selection (1×MHX=0.5:g/mlmycophenolic acid, 2.5: g/ml hypoxanthine, 50:g/ml xanthine) was addedand the plates incubated for an additional 2 to 3 weeks while coloniesformed.

Cell supernatants collected from wells with colonies were assayed forhuman IgG by ELISA as described. In brief, varying dilutions of the cellsupernatants were incubated in 96-well EIA plates coated with polyclonalgoat anti-human IgG Fc fragment and then bound human IgG was detectedusing Alkaline Phosphatase-conjugated goat anti-human IgG(H+L) and theappropriate color substrates. Standard curves, which used as standardthe same purified mAb that was being measured in the cell supernatants,were included on each EIA plate to enable quantitation of the human IgGin the supernatants. Cells in those colonies that appeared to beproducing the most human IgG were passaged into 24-well plates foradditional production determinations in spent cultures and thehighest-producing parental clones were subsequently identified.

The highest-producing parental clones were subcloned to identifyhigher-producing subclones and to prepare a more homogenous cell line.96-well tissue culture plates were seeded with one cell per well or fourcells per well in of IMDM, 5% FBS, 2 mM glutamine, 1×MHX and incubatedat 37° C. in a 5% CO₂ incubator for 12 to 20 days until colonies wereapparent. Cell supernatants were collected from wells that contained onecolony per well and analyzed by ELISA as described above. Selectedcolonies were passaged to 24-well plates and the cultures allowed to gospent before identifying the highest-producing subclones by quantitatingthe human IgG levels in their supernatants. This process was repeatedwhen selected first-round subclones were subjected to a second round ofsubcloning. The best second-round subclones were selected as the celllines for development.

Characterization of Cell Subclones. The best second-round subclones werechosen and growth curves performed to evaluate mAb production levels andcell growth characteristics. T75 flasks were seeded with 1×10⁵ cells/mlin 30 ml IMDM, 5% FBS, 2 mM glutamine, and 1×MHX (or serum-free media).Aliquots of 300 μl were taken at 24 hr intervals and live cell densitydetermined. The analyses continued until the number of live cells wasless than 1×10⁵ cells/ml. The collected aliquots of cell supernatantswere assayed for the concentration of antibody present. ELISA assayswere performed using as standard rTNV148B or rTNV14 JG92399. Sampleswere incubated for 1 hour on ELISA plates coated with polyclonal goatanti-human IgG Fc and bound mAb detected with AlkalinePhosphatase-conjugated goat anti-human IgG(H+L) at a 1:1000 dilution.

A different growth curve analysis was also done for two cell lines forthe purpose of comparing growth rates in the presence of varying amountsof MHX selection. Cell lines C466A and C466B were thawed into MHX-freemedia (IMDM, 5% FBS, 2 mM glutamine) and cultured for two additionaldays. Both cell cultures were then divided into three cultures thatcontained either no MHX, 0.2× MHX, or 1× MHX (1× MHX=0.5:g/mlmycophenolic acid, 2.5:g/ml hypoxanthine, 50:g/ml xanthine). One daylater, fresh T75 flasks were seeded with the cultures at a startingdensity of 1×10⁵ cells/ml and cells counted at 24 hour intervals for oneweek. Aliquots for mAb production were not collected. Doubling timeswere calculated for these samples using the formula provided in SOPPD32.025.

Additional studies were performed to evaluate stability of mAbproduction over time. Cultures were grown in 24-well plates in IMDM, 5%FBS, 2 mM glutamine, either with or without MHX selection. Cultures weresplit into fresh cultures whenever they became confluent and the olderculture was then allowed to go spent. At this time, an aliquot ofsupernatant was taken and stored at 4° C. Aliquots were taken over a55-78 day period. At the end of this period, supernatants were testedfor amount of antibody present by the anti-human IgG Fc ELISA asoutlined above.

Results and Discussion.

Inhibition of TNF binding to Recombinant Receptor.

A simple binding assay was done to determine whether the eight TNV mAbscontained in hybridoma cell supernatant were capable of blocking TNFαbinding to receptor. The concentrations of the TNV mAbs in theirrespective cell supernatants were first determined by standard ELISAanalysis for human IgG. A recombinant p55 TNF receptor/IgG fusionprotein, p55-sf2, was then coated on EIA plates and ¹²⁵I-labeled TNFαallowed to bind to the p55 receptor in the presence of varying amountsof TNV mAbs. As shown in FIG. 1, all but one (TNV122) of the eight TNVmAbs efficiently blocked TNFα binding to p55 receptor. In fact, the TNVmAbs appeared to be more effective at inhibiting TNFα binding than cA2positive control mAb that had been spiked into negative controlhybridoma supernatant. These results were interpreted as indicating thatit was highly likely that the TNV mAbs would block TNFα bioactivity incell-based assays and in vivo and therefore additional analyses werewarranted.

DNA Sequence Analysis.

Confirmation that the RNAs Encode Human mAbs.

As a first step in characterizing the seven TNV mAbs (TNV14, TNV15,TNV32, TNV86, TNV118, TNV148, and TNV196) that showed TNFα-blockingactivity in the receptor binding assay, total RNA was isolated from theseven hybridoma cell lines that produce these mAbs. Each RNA sample wasthen used to prepare human antibody heavy or light chain cDNA thatincluded the complete signal sequence, the complete variable regionsequence, and part of the constant region sequence for each mAb. ThesecDNA products were then amplified in PCR reactions and the PCR-amplifiedDNA was directly sequenced without first cloning the fragments. Theheavy chain cDNAs sequenced were >90% identical to one of the five humangermline genes present in the mice, DP-46 (FIG. 2). Similarly, the lightchain cDNAs sequenced were either 100% or 98% identical to one of thehuman germline genes present in the mice (FIG. 3). These sequenceresults confirmed that the RNA molecules that were transcribed into cDNAand sequenced encoded human antibody heavy chains and human antibodylight chains. It should be noted that, because the variable regions werePCR-amplified using oligonucleotides that map to the 5′ end of thesignal sequence coding sequence, the first few amino acids of the signalsequence may not be the actual sequence of the original TNV translationproducts but they do represent the actual sequences of the recombinantTNV mAbs.

Unique Neutralizing mAbs.

Analyses of the cDNA sequences for the entire variable regions of bothheavy and light chains for each mAb revealed that TNV32 is identical toTNV15, TNV118 is identical to TNV14, and TNV86 is identical to TNV148.The results of the receptor binding assay were consistent with the DNAsequence analyses, i.e. both TNV86 and TNV148 were approximately 4-foldbetter than both TNV118 and TNV14 at blocking TNF binding. Subsequentwork was therefore focused on only the four unique TNV mAbs, TNV14,TNV15, TNV148, and TNV196.

Relatedness of the Four mAbs

The DNA sequence results revealed that the genes encoding the heavychains of the four TNV mAbs were all highly homologous to each other andappear to have all derived from the same germline gene, DP-46 (FIG. 2).In addition, because each of the heavy chain CDR3 sequences are sosimilar and of the same length, and because they all use the J6 exon,they apparently arose from a single VDJ gene rearrangement event thatwas then followed by somatic changes that made each mAb unique. DNAsequence analyses revealed that there were only two distinct light chaingenes among the four mAbs (FIG. 3). The light chain variable regioncoding sequences in TNV14 and TNV15 are identical to each other and to arepresentative germline sequence of the Vg/38K family of human kappachains. The TNV148 and TNV196 light chain coding sequences are identicalto each other but differ from the germline sequence at two nucleotidepositions (FIG. 3).

The deduced amino acid sequences of the four mAbs revealed therelatedness of the actual mAbs. The four mAbs contain four distinctheavy chains (FIG. 4) but only two distinct light chains (FIG. 5).Differences between the TNV mAb sequences and the germline sequenceswere mostly confined to CDR domains but three of the mAb heavy chainsalso differed from the germline sequence in the framework regions (FIG.4). Compared to the DP-46 germline-encoded Ab framework regions, TNV14was identical, TNV15 differed by one amino acid, TNV148 differed by twoamino acids, and TNV196 differed by three amino acids.

Cloning of cDNAs, Site-specific Mutagenesis, and Assembly of FinalExpression Plasmids. Cloning of cDNAs. Based on the DNA sequence of thePCR-amplified variable regions, new oligonucleotides were ordered toperform another round of PCR amplification for the purpose of adaptingthe coding sequence to be cloned into expression vectors. In the case ofthe heavy chains, the products of this second round of PCR were digestedwith restriction enzymes BsiWI and BstBI and cloned into plasmid vectorL28 (plasmid identification numbers shown in Table 2). In the case ofthe light chains, the second-round PCR products were digested with SalIand AflII and cloned into plasmid vector pBC. Individual clones werethen sequenced to confirm that their sequences were identical to theprevious sequence obtained from direct sequencing of PCR products, whichreveals the most abundant nucleotide at each position in a potentiallyheterogeneous population of molecules.

Site-specific Mutagenesis to Change TNV148. mAbs TNV148 and TNV196 werebeing consistently observed to be four-fold more potent than the nextbest mAb (TNV14) at neutralizing TNFα bioactivity. However, as describedabove, the TNV148 and TNV196 heavy chain framework sequences differedfrom the germline framework sequences. A comparison of the TNV148 heavychain sequence to other human antibodies indicated that numerous otherhuman mAbs contained an Ile residue at position 28 in framework 1(counting mature sequence only) whereas the Pro residue at position 75in framework 3 was an unusual amino acid at that position.

A similar comparison of the TNV196 heavy chain suggested that the threeamino acids by which it differs from the germline sequence in framework3 may be rare in human mAbs. There was a possibility that thesedifferences may render TNV148 and TNV196 immunogenic if administered tohumans. Because TNV148 had only one amino acid residue of concern andthis residue was believed to be unimportant for TNFα binding, asite-specific mutagenesis technique was used to change a singlenucleotide in the TNV148 heavy chain coding sequence (in plasmid p1753)so that a germline Ser residue would be encoded in place of the Proresidue at position 75. The resulting plasmid was termed p1760 (seeTable 2). The resulting gene and mAb were termed TNV148B to distinguishit from the original TNV148 gene and mAb (see FIG. 5).

Assembly of Final Expression Plasmids. New antibody expression vectorswere prepared that were based on the 12B75 heavy chain and light chaingenes previously cloned as genomic fragments. Although different TNVexpression plasmids were prepared (see Table 2), in each case the 5′flanking sequences, promoter, and intron enhancer derived from therespective 12B75 genes. For the light chain expression plasmids, thecomplete J-C intron, constant region coding sequence and 3′ flankingsequence were also derived from the 12B75 light chain gene. For theheavy chain expression plasmids that resulted in the final productioncell lines (p1781 and p1783, see below), the human IgG1 constant regioncoding sequences derived from Centocor's previously-used expressionvector (p104). Importantly, the final production cell lines reportedhere express a different allotype (Gm(f+)) of the TNV mAbs than theoriginal, hybridoma-derived TNV mAbs (G1m(z)). This is because the 12B75heavy chain gene derived from the GenPharm mice encodes an Arg residueat the C-terminal end of the CH1 domain whereas Centocor's IgG1expression vector p104 encodes a Lys residue at that position. Otherheavy chain expression plasmids (e.g. p1786 and p1788) were prepared inwhich the J-C intron, complete constant region coding sequence and 3′flanking sequence were derived from the 12B75 heavy chain gene, but celllines transfected with those genes were not selected as the productioncell lines. Vectors were carefully designed to permit one-step cloningof future PCR-amplified V regions that would result in final expressionplasmids.

PCR-amplified variable region cDNAs were transferred from L28 or pBCvectors to intermediate-stage, 12B75-based vectors that provided thepromoter region and part of the J-C intron (see Table 2 for plasmididentification numbers). Restriction fragments that contained the 5′half of the antibody genes were then transferred from theseintermediate-stage vectors to the final expression vectors that providedthe 3′ half of the respective genes to form the final expressionplasmids (see Table 2 for plasmid identification numbers).

Cell Transfections and Subcloning. Expression plasmids were eitherlinearized by restriction digest or the antibody gene inserts in eachplasmid were purified away from the plasmid backbones. Sp2/0 and 653mouse myeloma cells were transfected with the heavy and light chain DNAby electroporation. Fifteen different transfections were done, most ofwhich were unique as defined by the Ab, specific characteristics of theAb genes, whether the genes were on linearized whole plasmids orpurified gene inserts, and the host cell line (summarized in Table 4).Cell supernatants from clones resistant to mycophenolic acid wereassayed for the presence of human IgG by ELISA and quantitated usingpurified rTNV148B as a reference standard curve.

Highest-producing rTNV148B Cell Lines

Ten of the best-producing 653 parental lines from rTNV148B transfection2 (produced 5-10:g/ml in spent 24-well cultures) were subcloned toscreen for higher-producing cell lines and to prepare a more homogeneouscell population. Two of the subclones of the parental line 2.320,2.320-17 and 2.320-20, produced approximately 50:g/ml in spent 24-wellcultures, which was a 5-fold increase over their parental line. A secondround of subcloning of subcloned lines 2.320-17 and 2.320-20 led

The identification numbers of the heavy and light chain plasmids thatencode each mAb are shown. In the case of transfections done withpurified mAb gene inserts, plasmid p13 (pSV2gpt) was included as asource of the gpt selectable marker. The heavy chain constant regionswere encoded either by the same human IgG1 expression vector used toencode Remicade (‘old’) or by the constant regions contained within the12B75 (GenPharm/Medarex) heavy chain gene (‘new’). H1/L2 refers to a“novel” mAb made up of the TNV14 heavy chain and the TNV148 light chain.Plasmids p1783 and p1801 differ only by how much of the J-C intron theirheavy chain genes contain. The transfection numbers, which define thefirst number of the generic names for cell clones, are shown on theright. The rTNV148B-producing cell lines C466 (A, B, C, D) and C467Adescribed here derived from transfection number 2 and 1, respectively.The rTNV14-producing cell line C476A derived from transfection number 3.

TABLE 4 Summary of Cell Transfections. Plasmids HC DNA Transfection no.mAb HC/LC/gpt vector format Sp2/0 653 rTNV148B 1783/1776 old linear  1 2 rTNV14 1781/1775 old linear  3 — rTNV148B 1788/1776/13 new insert 4,6 5, 7 rTNV14 1786/1775/13 new insert  8, 10  9, 11 rTNV148 1787/1776/13new insert 12 17 rH1/L2 1786/1776/13 new insert 13 14 rTNV148B 1801/1776old linear 16

ELISA assays on spent 24-well culture supernatants indicated that thesesecond-round subclones all produced between 98 and 124:g/ml, which wasat least a 2-fold increase over the first-round subclones. These 653cell lines were assigned C code designations as shown in Table 5.

Three of the best-producing Sp2/0 parental lines from rTNV148Btransfection 1 were subcloned. Two rounds of subcloning of parental line1.73 led to the identification of a clone that produced 25:g/ml in spent24-well cultures. This Sp2/0 cell line was designated C467A (Table 5).

Highest-producing rTNV14 Cell Lines

Three of the best-producing Sp2/0 parental lines from rTNV14transfection 3 were subcloned once. Subclone 3.27-1 was found to be thehighest-producer in spent 24-well cultures with a production of 19:g/ml.This cell line was designated C476A (Table 5).

TABLE 5 Summary of Selected Production Cell Lines and their C codes. Thefirst digit of the original clone names indicates which transfection thecell line derived from. All of the C-coded cell lines reported here werederived from transfections with heavy and light chain whole plasmidsthat had been linearized with restriction enzymes. Original Spent24-well mAb Clone Name C code Host Cell Production rTNV148B 2.320-17-36C466A 653 103: g/ml 2.320-20-111 C466B 653 102: g/ml 2.320-17-4 C466C653  98: g/ml 2.320-20-99 C466D 653 124: g/ml 1.73-12-122 C467A Sp2/0 25: g/ml rTNV14 3.27-1 C476A Sp2/0  19: g/ml

Characterization of Subcloned Cell Lines

To more carefully characterize cell line growth characteristics anddetermine mAb-production levels on a larger scale, growth curvesanalyses were performed using T75 cultures. The results showed that eachof the four C466 series of cell lines reached peak cell density between1.0×10⁶ and 1.25×10⁶ cells/ml and maximal mAb accumulation levels ofbetween 110 and 140:g/ml (FIG. 7). In contrast, the best-producing Sp2/0subclone, C467A, reached peak cell density of 2.0×10⁶ cells/ml andmaximal mAb accumulation levels of 25:g/ml (FIG. 7). A growth curveanalysis was not done on the rTNV14-producing cell line, C476A.

An additional growth curve analysis was done to compare the growth ratesin different concentrations of MHX selection. This comparison wasprompted by recent observations that C466 cells cultured in the absenceof MHX seemed to be growing faster than the same cells cultured in thenormal amount of MHX (1×). Because the cytotoxic concentrations ofcompounds such as mycophenolic acid tend to be measured over orders ofmagnitude, it was considered possible that the use of a lowerconcentration of MHX might result in significantly faster cell doublingtimes without sacrificing stability of mAb production. Cell lines C466Aand C466B were cultured either in: no MHX, 0.2× MHX, or 1× MHX. Livecell counts were taken at 24-hour intervals for 7 days. The results didreveal an MHX concentration-dependent rate of cell growth (FIG. 8). Cellline C466A showed a doubling time of 25.0 hours in 1× MHX but only 20.7hours in no MHX. Similarly, cell line C466B showed a doubling time of32.4 hours in 1× MHX but only 22.9 hours in no MHX. Importantly, thedoubling times for both cell lines in 0.2× MHX were more similar to whatwas observed in no MHX than in 1× MHX (FIG. 8). This observation raisesthe possibility than enhanced cell performance in bioreactors, for whichdoubling times are an important parameter, could be realized by usingless MHX. However, although stability test results (see below) suggestthat cell line C466D is capable of stably producing rTNV148B for atleast 60 days even with no MHX present, the stability test also showedhigher mAb production levels when the cells were cultured in thepresence of MHX compared to the absence of MHX.

To evaluate mAb production from the various cell lines over a period ofapproximately 60 days, stability tests were performed on cultures thateither contained, or did not contain, MHX selection. Not all of the celllines maintained high mAb production. After just two weeks of culture,clone C466A was producing approximately 45% less than at the beginningof the study. Production from clone C466B also appeared to dropsignificantly. However, clones C466C and C466D maintained fairly stableproduction, with C466D showing the highest absolute production levels(FIG. 9).

Conclusion

From an initial panel of eight human mAbs against human TNFα, TNV148Bwas selected as preferred based on several criteria that includedprotein sequence and TNF neutralization potency, as well as TNV14. Celllines were prepared that produce greater than 100:g/ml of rTNV148B and19:g/ml rTNV14.

Example 5: Arthritic Mice Study Using Anti-TNF Antibodies and ControlsUsing

Single Bolus Injection

At approximately 4 weeks of age the Tg197 study mice were assigned,based on gender and body weight, to one of 9 treatment groups andtreated with a single intraperitoneal bolus dose of Dulbecco's PBS(D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148or TNV196) at either 1 mg/kg or 10 mg/kg.

RESULTS: When the weights were analyzed as a change from pre-dose, theanimals treated with 10 mg/kg cA2 showed consistently higher weight gainthan the D-PBS-treated animals throughout the study. This weight gainwas significant at weeks 3-7. The animals treated with 10 mg/kg TNV148also achieved significant weight gain at week 7 of the study. (See FIG.10).

FIGS. 11A-C represent the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index waslower than the D-PBS control group starting at week 3 and continuingthroughout the remainder of the study (week 7). The animals treated with1 mg/kg TNV14 and the animals treated with 1 mg/kg cA2 failed to showsignificant reduction in AI after week 3 when compared to theD-PBS-treated Group. There were no significant differences between the10 mg/kg treatment groups when each was compared to the others ofsimilar dose (10 mg/kg cA2 compared to 10 mg/kg TNV14, 148 and 196).When the 1 mg/kg treatment groups were compared, the 1 mg/kg TNV148showed a significantly lower AI than 1 mg/kg cA2 at 3, 4 and 7 weeks.The 1 mg/kg TNV148 was also significantly lower than the 1 mg/kgTNV14-treated Group at 3 and 4 weeks. Although TNV196 showed significantreduction in AI up to week 6 of the study (when compared to theD-PBS-treated Group), TNV148 was the only 1 mg/kg treatment thatremained significant at the conclusion of the study.

Example 6: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Multiple Bolus Doses

At approximately 4 weeks of age the Tg197 study mice were assigned,based on body weight, to one of 8 treatment groups and treated with aintraperitoneal bolus dose of control article (D-PBS) or antibody(TNV14, TNV148) at 3 mg/kg (week 0). Injections were repeated in allanimals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for testarticle efficacy. Serum samples, obtained from animals in Groups 7 and 8were evaluated for immune response induction and pharmacokineticclearance of TNV14 or TNV148 at weeks 2, 3 and 4.

RESULTS: No significant differences were noted when the weights wereanalyzed as a change from pre-dose. The animals treated with 10 mg/kgcA2 showed consistently higher weight gain than the D-PBS-treatedanimals throughout the study. (See FIG. 12).

FIGS. 13A-C represent the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index wassignificantly lower than the D-PBS control group starting at week 2 andcontinuing throughout the remainder of the study (week 5). The animalstreated with 1 mg/kg or 3 mg/kg of cA2 and the animals treated with 3mg/kg TNV14 failed to achieve any significant reduction in AI at anytime throughout the study when compared to the d-PBS control group. Theanimals treated with 3 mg/kg TNV148 showed a significant reduction whencompared to the d-PBS-treated group starting at week 3 and continuingthrough week 5. The 10 mg/kg cA2-treated animals showed a significantreduction in AI when compared to both the lower doses (1 mg/kg and 3mg/kg) of cA2 at weeks 4 and 5 of the study and was also significantlylower than the TNV14-treated animals at weeks 3-5. Although thereappeared to be no significant differences between any of the 3 mg/kgtreatment groups, the AI for the animals treated with 3 mg/kg TNV14 weresignificantly higher at some time points than the 10 mg/kg whereas theanimals treated with TNV148 were not significantly different from theanimals treated with 10 mg/kg of cA2.

Example 7: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Single Intraperitoneal Bolus Dose

At approximately 4 weeks of age the Tg197 study mice were assigned,based on gender and body weight, to one of 6 treatment groups andtreated with a single intraperitoneal bolus dose of antibody (cA2, orTNV148) at either 3 mg/kg or 5 mg/kg. This study utilized the D-PBS and10 mg/kg cA2 control Groups.

When the weights were analyzed as a change from pre-dose, all treatmentsachieved similar weight gains. The animals treated with either 3 or 5mg/kg TNV148 or 5 mg/kg cA2 gained a significant amount of weight earlyin the study (at weeks 2 and 3). Only the animals treated with TNV148maintained significant weight gain in the later time points. Both the 3and 5 mg/kg TNV148-treated animals showed significance at 7 weeks andthe 3 mg/kg TNV148 animals were still significantly elevated at 8 weekspost injection. (See FIG. 14).

FIG. 15 represents the progression of disease severity based on thearthritic index. All treatment groups showed some protection at theearlier time points, with the 5 mg/kg cA2 and the 5 mg/kg TNV148 showingsignificant reductions in AI at weeks 1-3 and all treatment groupsshowing a significant reduction at week 2. Later in the study theanimals treated with 5 mg/kg cA2 showed some protection, withsignificant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) ofboth the cA2 and the TNV148 showed significant reductions at 6 and alltreatment groups showed significant reductions at week 7. None of thetreatment groups were able to maintain a significant reduction at theconclusion of the study (week 8). There were no significant differencesbetween any of the treatment groups (excluding the saline control group)at any time point.

Example 8: Arthritic Mice Study Using Anti-TNF Antibodies and Controlsas Single Intraperitoneal Bolus Dose Between Anti-TNF Antibody andModified Anti-TNF Antibody

To compare the efficacy of a single intraperitoneal dose of TNV148(derived from hybridoma cells) and rTNV148B (derived from transfectedcells). At approximately 4 weeks of age the Tg197 study mice wereassigned, based on gender and body weight, to one of 9 treatment groupsand treated with a single intraperitoneal bolus dose of Dulbecco=S PBS(D-PBS) or antibody (TNV148, rTNV148B) at 1 mg/kg.

When the weights were analyzed as a change from pre-dose, the animalstreated with 10 mg/kg cA2 showed a consistently higher weight gain thanthe D-PBS-treated animals throughout the study. This weight gain wassignificant at weeks 1 and weeks 3-8. The animals treated with 1 mg/kgTNV148 also achieved significant weight gain at weeks 5, 6 and 8 of thestudy. (See FIG. 16).

FIG. 17 represents the progression of disease severity based on thearthritic index. The 10 mg/kg cA2-treated group's arthritic index waslower than the D-PBS control group starting at week 4 and continuingthroughout the remainder of the study (week 8). Both of theTNV148-treated Groups and the 1 mg/kg cA2-treated Group showed asignificant reduction in AI at week 4. Although a previous study(P-099-017) showed that TNV148 was slightly more effective at reducingthe Arthritic Index following a single 1 mg/kg intraperitoneal bolus,this study showed that the AI from both versions of the TNVantibody-treated groups was slightly higher. Although (with theexception of week 6) the 1 mg/kg cA2-treated Group was not significantlyincreased when compared to the 10 mg/kg cA2 group and the TNV148-treatedGroups were significantly higher at weeks 7 and 8, there were nosignificant differences in AI between the 1 mg/kg cA2, 1 mg/kg TNV148and 1 mg/kg TNV148B at any point in the study.

Example 9: Anti-TNF Antibody for the Treatment or Prevention ofAnkylosing Spondylitis Synopsis

A Multicenter, Randomized, Double-blind, Placebo-controlled Trial ofGolimumab, an Anti-TNFα Monoclonal Antibody, Administered Intravenously,in Subjects with Active Ankylosing Spondylitis.

SIMPONI® (golimumab) is a fully human monoclonal antibody with anImmunoglobulin G 1 (IgG1) heavy chain isotype (G1m[z] allotype) and akappa light chain isotype. Golimumab has a heavy chain (HC) comprisingSEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37. Themolecular weight of golimumab ranges from 149,802 to 151,064 daltons.Golimumab binds to human tumor necrosis factor alpha (TNFα) with highaffinity and specificity and neutralizes TNFα bioactivity.

Objectives and Hypothesis

Primary Objective

The primary objective of this study is to evaluate the efficacy of IVadministration of golimumab 2 mg/kg in subjects with active ankylosingspondylitis (AS) by assessing the reduction in signs and symptoms of AS.

Secondary Objectives

The secondary objectives are to assess the following for golimumab:

Efficacy related to improving physical function, range of motion,health-related quality of life, and other health outcomes

Safety

Pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity

Hypothesis

To address the primary objective of the study, the statisticalhypothesis (alternative hypothesis) is that IV golimumab 2 mg/kg isstatistically superior to placebo in reducing the signs and symptoms ofsubjects with active AS based on the primary efficacy endpoint.

The primary endpoint of this study is the proportion of subjects whoachieve a 20% improvement from baseline in the ASsessment in AnkylosingSpondylitis (ASAS) International Working Group criteria (called ASAS20)at Week 16. This endpoint was chosen because it is well-accepted byregulatory authorities and the clinical AS community.

Overview of Study Design

This is a Phase 3 multicenter, randomized, double-blind,placebo-controlled study of the efficacy and safety of IV golimumabcompared with placebo in subjects with active AS with an inadequateresponse or intolerance to NSAIDs. Approximately 200 subjects will berandomized at approximately 40 investigational sites. All subjects willbe randomly assigned to receive golimumab 2 mg/kg or placebo IVinfusions at Weeks 0, 4, and every 8 weeks (q8w) thereafter through Week52. At Week 16, all subjects receiving placebo infusions will cross-overand begin to receive golimumab IV infusions.

Subjects in the golimumab IV treatment group will continue to receivegolimumab IV infusions. Database locks are scheduled for Weeks 28 and60. Subjects will be followed for adverse events (AE) and seriousadverse events (SAE) at least 8 weeks following the last study treatmentadministration. The end of study is defined as the time the last subjectcompletes the Week 60 visit.

Subject Population

Subjects eligible for the study will be men or women 18 years of age orolder with a diagnosis of AS for at least 3 months defined as “definite”by the modified New York criteria, and symptoms of active disease, asevidenced by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)≥4 and a Visual Analogue Scale (VAS) for total back pain of ≥4, each ona scale of 0 to 10 cm. Subjects are required to have a C-reactiveprotein (CRP) level of ≥0.3 mg/dL.

Other major features of the study population are as follows:

Current users of methotrexate (MTX), sulfasalazine (SSZ), andhydroxychloroquine (HCQ) and low dose oral corticosteroids are permittedand should enter the study on stable doses of these medications.

Subjects with prior exposure to no more than one biologic anti-TNFαagent (other than golimumab) are permitted to be included in the study,but will be limited to at most 20% of the study population.

Subjects with complete ankylosis of the spine, defined as bridgingsyndesmophytes present at all intervertebral levels of the cervical andlumbar spine visualized on lateral-view spinal radiographs are permittedto be included in the study, but will be limited to at most 10% of thestudy population.

Screening for eligible subjects will be performed within 6 weeks beforeadministration of the study agent.

Subjects must also meet the inclusion and exclusion criteria.

Dosage and Administration

At the initial screening visit, informed consent will be obtained fromall subjects who are deemed potentially eligible for the study,according to the protocol-specified inclusion and exclusion criteria,for enrollment in the study. At the randomization visit, subjects willbe re-assessed and, if all specified inclusion and exclusion criteriaare met, subjects will be randomized to receive either golimumab IVinfusions or placebo IV infusions. Randomization will be stratified bygeographic region and prior use of anti-TNFα therapy.

Before the first study infusion, subjects will be randomly assigned in a1:1 ratio to 1 of the following 2 treatment groups:

Group 1 (n=100): Subjects will receive IV placebo infusions at Weeks 0,4, and 12. Subjects will cross over to IV golimumab 2 mg/kg at Weeks 16,and receive administrations at Weeks 16, 20, and q8w thereafter.

Group 2 (n=100): Subjects will receive 2 mg/kg of IV golimumab at Weeks0, 4, and q8w thereafter. Subjects will receive an IV placebo infusionat Week 16 to maintain the blind.

All infusions will be completed over 30±10 minutes.

Efficacy Evaluations/Endpoints

Efficacy evaluations chosen for this study were established in previoustrials of therapeutic biologic agents for the treatment of AS. Patientreported outcomes (PRO) chosen for this study are consistent withclinically relevant measurements that are accepted in the medicalliterature for other studies in AS and applicable US/EU regulatoryguidance documents.

Ankylosing spondylitis response evaluations include:

Bath Ankylosing Spondylitis Functional Index (BASFI)

Patient's Global Assessment

Total Back Pain

Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)

36-item short form health survey (SF-36)

Bath Ankylosing Spondylitis Metrology Index (BASMI)

Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire

Chest Expansion

Night Back Pain

Enthesitis Index

Medical Outcomes Study Sleep scale

Work Limitations Questionnaire (WLQ)

Productivity Visual Analog Scale

EuroQol-5D (EQ-5D) Questionnaire

Primary Endpoint

The primary endpoint of this study is the proportion of ASAS 20responders at Week 16. The study will be considered positive if theproportion of subjects with ASAS 20 at Week 16 is demonstrated to bestatistically significantly greater in the golimumab group compared withthe placebo group.

Major Secondary Endpoints

The following major secondary analyses will be performed. The endpointsare listed in order of importance as specified below:

1. The proportion of subjects who achieve an ASAS 40 at Week 16.

2. The proportion of subjects who achieve at least 50% improvement frombaseline in BASDAI at Week 16.

3. The change from baseline in BASFI at Week 16.

Pharmacokinetic Evaluations

Blood samples will be collected at selected visits to evaluate the PK ofIV golimumab in adult subjects with AS. Pharmacokinetics samples shouldbe drawn from a different arm than the IV infusion line if study agentis administered at that visit. Specifically, at the visits at Weeks 0,4, 12, 20, 36, and 52, 2 samples for serum golimumab concentrations willbe collected: 1 sample will be collected immediately prior to infusionand the other collected one hour after the end of the infusion. For eachof the remaining visits, only 1 sample for serum golimumabconcentrations will be collected. This sample should be collected priorto infusion if an infusion of the study agent is administered at thatvisit. A random PK sample will also be drawn for population PK analysisbetween the Weeks 12 and 20 visits other than at the time of the Week12, Week 16, or Week 20 visit; this sample must be collected at least 24hours prior to or after a study agent infusion. At applicable timepoints, sera for the measurement of both golimumab concentration andantibodies to golimumab will be derived from the same blood draw.

Immunogenicity Evaluations

To evaluate the immunogenicity of golimumab in adult subjects with AS,serum samples for the detection of antibodies to golimumab will becollected according to the Time and Events Schedule.

Biomarker Evaluations

Biomarker samples will be collected to gain a molecular understanding ofinter-individual variability in clinical outcomes, which may help toidentify population subgroups that respond differently to the drug. Thebiomarker samples may also be used to help address emerging issues andto enable the development of safer, more effective, and ultimatelyindividualized therapies in the future.

Pharmacogenomics (DNA) Evaluations

Genomic testing will be done to search for links of specific genes todisease or response to drug. Only DNA research related to golimumab orto the diseases for which this drug was developed will be performed.Genome wide pharmacogenomics and/or epigenetics testing will beundertaken in this study in consenting subjects. Subjects participatingin this portion of the study must sign a separate informed consent.Further, a subject may withdraw such consent at any time withoutaffecting their participation in other aspects of the study, or theirfuture participation in the study.

A pharmacogenomics blood sample will be collected to allow forpharmacogenomics research, as necessary (where local regulationspermit). Subject participation in the pharmacogenomics research isoptional.

Safety Evaluations

Based upon the safety profile of other anti-TNFα agents, as well as thegolimumab safety data to date, several AEs of interest have beenidentified and will be monitored and assessed in this study. Theseinclude: infusion reactions, hepatobiliary laboratory abnormalities,infections including TB, and malignancies.

Statistical Methods

Simple descriptive summary statistics, such as n, mean, SD, median, IQrange, minimum, and maximum for continuous variables, and counts andpercentages for discrete variables will be used to summarize most data.

The Cochran-Mantel-Haenszel (CMH) test stratified by prior use ofanti-TNFα therapy will be used to compare categorical variables such asthe proportion of subjects responding to treatment. In general, ANOVAwith prior use of anti-TNFα therapy as a factor will be used foranalyzing continuous variables, unless otherwise stated. All statisticaltests will be performed at α=0.05 (2-sided). In addition to statisticalanalyses, graphical data displays (egg, line plots) and subject listingsmay also be used to summarize/present the data.

Population Set

The population set will be an intent-to-treat population (i.e., allrandomized subjects). Subjects included in the efficacy analyses will besummarized according to their assigned treatment group regardless ofwhether or not they receive the assigned treatment.

Safety and PK analyses will include all subjects who received at leastone administration of study treatment.

Endpoint Analyses

Primary Endpoint Analysis

To address the primary objective, the proportion of subjects with ASAS20 response at Week 16 (primary endpoint) will be compared between theplacebo and golimumab groups using a CMH test stratified by prior use ofanti-TNFα therapy (Yes/No) at a significance level of 0.05 (2-sided). Inthis primary efficacy analysis, data from all randomized subjects willbe analyzed according to their assigned treatment group regardless oftheir actual treatment received. A last observation carried forward(LOCF) procedure will be used to impute the missing ASAS components ifthe subjects have data for at least 1 ASAS component at Week 16. If thesubjects do not have data for all the ASAS components at Week 16, thesubjects will be considered non-responders.

Major Secondary Endpoint Analyses

The following major secondary analyses will be performed in order ofimportance as specified below:

1. The proportion of subjects who achieve an ASAS 40 at Week 16 will becompared between treatment groups.

2. The proportion of subjects who achieve at least 50% improvement frombaseline in BASDAI at Week 16 will be compared between treatment groups.

3. The change from baseline in BASFI at Week 16 will be compared betweentreatment groups.

To control the Type I error rate for multiplicity, the first majorsecondary endpoint will be tested only if the primary endpoint achievedstatistical significance at a 0.05 level of significance (2-sided). Thesubsequent major secondary endpoints will be tested only if the primaryendpoint and the preceding major secondary endpoint(s) are statisticallysignificant at a 0.05 level of significance (2-sided).

Safety Analysis Overview

Routine safety evaluations will be performed. The occurrences and typeof AEs, SAEs, and reasonably related AEs including infusion reactionsand infections including

TB, will be summarized by treatment groups. The number of subjects withabnormal laboratory parameters (hematology and chemistry) based on NCICTCAE toxicity grading will be summarized. In addition, the number ofsubjects with ANA and anti-dsDNA antibodies and the relationship ofinfusion reactions with antibodies to golimumab will be summarized.

All safety analyses will be performed using the population of allsubjects who received at least 1 administration of study agent. Analyseswill be performed using the treatment that the subjects actuallyreceived.

In addition, graphical data displays (egg, line plots) and subjectlistings may also be used to summarize/present data.

ABBREVIATIONS

AE adverse event

AS ankylosing spondylitis

ASAS 20 Assessment in ankylosing spondylitis 20

ASQoL Ankylosing spondylitis quality of life

ASSERT Ankylosing Spondylitis Study for the Evaluation of RecombinantInfliximab Therapy

BASDAI Bath ankylosing spondylitis disease activity index

BASFI Bath ankylosing spondylitis functional index

BASMI Bath ankylosing spondylitis metrology index

BCG Bacille Calmette-Guérin

CHF congestive heart failure

CMH Cochran-Mantel-Haenszel

CRP c-reactive protein

DAS disease activity score

DBL data base lock

DMARD disease modifying antirheumatic drug

DMC Data Monitoring Committee

DNA deoxyribonucleic acid

ECG electrocardiogram

eCRF electronic case report form

eDC electronic data capture

EQ-5D EuroQol-5D

EQ VAS EQ visual analogue scale

EU European Union

GCP Good Clinical Practice

HBV hepatitis B virus

HCQ hydroxychloroquine

HCV hepatitis C virus

HIV human immunodeficiency virus

HRQOL Health-related quality of life

IB Investigator's Brochure

ICF informed consent form

ICH International Conference on Harmonisation

IEC independent ethics committee

IgG 1 immunoglobulin G 1

IMA independent musculoskeletal assessor

IRB Institutional Review Board

IV intravenous

IWRS interactive web response system

MCS mental component summary

MOS-SS Medical Outcomes Study Sleep Scale

MMP-1 matrix metalloproteinase-1

MMP-3 matrix metalloproteinase-3

MTX methotrexate

NSAID non-steroidal anti-inflammatory drug

PCS physical component summary

PD pharmacodynamics

PK pharmacokinetics

PQC product quality complaint

PRO patient reported outcomes

PsA psoriatic arthritis

q8w every 8 weeks

RA rheumatoid arthritis

RBC red blood cell

SAE serious adverse event

SAP statistical analysis plan

SC subcutaneous

SF-36 36-item short form health survey

SSZ sulfasalazine

TB tuberculosis

TNFα tumor necrosis factor alpha

TST tuberculin skin test

US United States

VAS visual analogue scale

WBC white blood cell

WLQ work limitations questionnaire

Introduction

Golimumab is a fully human monoclonal antibody with an Immunoglobulin G1 (IgG1) heavy chain isotype (Glm[z] allotype) and a kappa light chainisotype. Golimumab has a heavy chain (HC) comprising SEQ ID NO:36 and alight chain (LC) comprising SEQ ID NO:37. The molecular weight ofgolimumab ranges from 149,802 to 151,064 daltons. Golimumab is a humanmonoclonal antibody that forms high affinity, stable complexes with boththe soluble and transmembrane bioactive forms of human tumor necrosisfactor alpha (TNFα), which prevents the binding of TNFα to itsreceptors. No binding to other TNFα superfamily ligands was observed; inparticular, golimumab does not bind or neutralize human lymphotoxin.Tumor necrosis factor α is synthesized primarily by activated monocytes,macrophages and T cells as a transmembrane protein that self-associatesto form the bioactive homotrimer and is rapidly released from the cellsurface by proteolysis. The binding of TNFα to either the p55 or p75 TNFreceptors leads to the clustering of the receptor cytoplasmic domainsand initiates signaling. Tumor necrosis factor α has been identified asa key sentinel cytokine that is produced in response to various stimuliand subsequently promotes the inflammatory response through activationof the caspase-dependent apoptosis pathway and the transcription factorsnuclear factor (NF)-κB and activator protein-1 (AP-1). Tumor necrosisfactor α also modulates the immune response through its role in theorganization of immune cells in germinal centers. Elevated expression ofTNFα has been linked to chronic inflammatory diseases such as rheumatoidarthritis (RA), as well as spondyloarthropathies such as psoriaticarthritis (PsA) and ankylosing spondylitis (AS), and is an importantmediator of the articular inflammation and structural damage that arecharacteristic of these diseases.

Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknownetiology that involves the sacroiliac joints, and often the axialskeleton, entheses, and peripheral joints. AS affects men more oftenthan women and its prevalence in the United States is estimated at0.2-0.5% of the population.²² Chronic inflammation of entheses leads tonew bone formation, syndesmophytes, and ankylosis of joints, primarilyin the axial skeleton. It is this axial ankylosis that may lead todramatic loss of range of motion and to disability. The disease may alsohave extraskeletal manifestations, including uveitis, carditis,pulmonary fibrosis, bowel inflammation, and cardiac conductionabnormalities. Ankylosing spondylitis, considered a subset of thespondyloarthropathies, is strongly associated with the presence of thehuman leukocyte antigen- B27 (HLA-B27) antigen.³⁴

Although patients may experience a variety of musculoskeletal symptoms(proximal arthralgias, chest pain, and tenderness around peripheraljoints from enthesitis), the most common presenting symptom is chroniclow-back pain. The low-back pain usually begins before age 40, isinsidious in onset, associated with morning stiffness, and eventually,is symmetrical. These musculoskeletal symptoms may be associated withconstitutional symptoms, such as fatigue, fever, and weight loss. UntilTNF α inhibitors were approved, treatments for AS had limited efficacy,and consisted mainly of exercise and NSAIDs, with a role for oralsulfasalazine (SSZ) in the subset of patients with peripheralarthritis.¹⁰ Biologic TNF α inhibitors have been shown in randomizedcontrolled trials to significantly improve signs and symptoms, mobilityand physical function in patients with AS.

Role of TNFαi in Ankylosing Spondylitis

The efficacy and safety profile of anti-TNF α therapy for a variety ofindications, including AS, has been well-characterized. Tumor necrosisfactor α is considered a key inflammatory mediator that exhibits a widevariety of functional activities. Abnormally high levels of TNF α havebeen implicated in the pathophysiology of several immune-mediateddiseases, including RA, PsA, and AS. Binding of TNF α by an anti-TNF αantibody prevents the target from binding to cell surface TNF αreceptors, and consequently prevents downstream signaling cascades andthe deleterious effects of inappropriate or excessive TNF α expression.Elevated levels of TNF α have been observed in both peripheralblood^(16,20,26) and synovial tissue⁹ from patients with active AS. Ithas been suggested that TNF α probably plays a role in the sacroiliitisof AS as it does in the synovitis of RA. In a study of 5 patients withactive AS who were evaluated with computed tomography-directedsacroiliac joint biopsies, immunohistologic analysis revealed cellularinfiltrates consisting mostly of T cells and macrophages, and in situhybridization studies of 3 subjects' biopsies revealed abundant TNF α.⁵

A number of open-label and double-blind placebo-controlled trials haveshown the substantial efficacy of infliximab, a recombinant IgG1-κhuman-murine chimeric monoclonal anti-TNF α antibody, in alleviating thesigns and symptoms of AS. Infliximab was the first anti-TNF α agentstudied as a treatment for AS, and an induction regimen of 5 mg/kginfliximab infusions at 0, 2, and 6 weeks in subjects with either AS orsubjects diagnosed with spondyloarthropathy including AS resulted inrapid improvement in disease activity measures.^(3,7,24,25,27)

Two randomized, double-blind, placebo-controlled trials of infliximab inpatients with AS only⁶ and with spondyloarthropathy including AS²⁸showed that infliximab therapy resulted in rapid, significantimprovement in clinical outcome measures. In the ASSERT study (a large,multicenter, double-blind, placebo-controlled trial of infliximabinvolving 279 subjects with AS), ASsessment in Ankylosing Spondylitis 20(ASAS 20) response rates at 24 weeks were 60% in infliximab-treatedsubjects versus 18% in the placebo-treated group.²⁹ There was alsosignificant improvement in measures of physical function, range ofmotion quality of life, and in disease activity score on MRI. Infliximabtherapy in AS patients was generally well tolerated.

Tumor necrosis factor α inhibition in AS using subcutaneous (SC) drugs,including etanercept, adalimumab, golimumab, and certolizumab, also hasbeen shown to be efficacious in randomized, placebo-controlledtrials.^(4,11,18,19,30) While the precise role of TNF α in thepathophysiology of AS is yet unclear, there is already a large andmounting body of evidence that TNF α inhibition is of major therapeuticbenefit in this disease.

Overall Rationale for the Study

Although therapy with anti-TNFα agents has been used successfully in thetreatment of inflammatory arthritides, anti-TNFα agents have limitationswith respect to safety, dosing regimen, cost, and immunogenicity. Toaddress some of these limitations, a fully human anti-TNFα mAb,designated golimumab (also known as CNTO 148 and rTNV148B). Golimumab, afully human anti-TNFα mAb, binds with high affinity to human TNFα andinhibits TNFα bioactivity. In addition, golimumab inhibits TNFα-mediatedcell cytotoxicity and TNFα-mediated endothelial cell activation.Golimumab also induces activation of complement-mediated cell lysis andreduces the development of arthritis in mice that overexpress humanTNFα.

Treatment with anti-TNF α agents, including SC golimumab, has beendemonstrated to significantly improve signs and symptoms, physicalfunction, and health-related quality of life (HRQOL) in subjectsaffected by AS. A global, randomized, doubleblind, placebo-controlledPhase 3 study was completed for SC administration of golimumab insubjects with AS (Study C0524T09) to evaluate the long-term safety andefficacy of SC golimumab through 5 years of follow-up. Subcutaneousgolimumab was demonstrated to be efficacious in improving the signs andsymptoms of AS. Safety analyses showed that SC golimumab was generallywell tolerated, and demonstrated a safety profile similar to thatobserved with other anti-TNF a agents.

Given the known safety and efficacy of SC golimumab, it was anticipatedthat IV golimumab would prove efficacious with an acceptable safetyprofile consistent with other anti-TNF α agents in rheumatologicdiseases such as RA, PsA, and AS. Intravenous golimumab has beendefinitively studied in a Phase 3 study (CNTO148ART3001) that formed thebasis of approval for the treatment of RA. The CNTO148ART3001 study wasa randomized, double-blind, placebo-controlled, multicenter, 2-arm studyof the efficacy and safety of IV administration of golimumab 2 mg/kginfusions administered over a period of 30±10 minutes at Weeks 0, 4, andevery 8 weeks (q8w) thereafter in subjects with active RA despiteconcurrent methotrexate (MTX) therapy. Subjects with active RA despiteMTX were randomized to receive either placebo infusions or IV golimumabadministered 2 mg/kg at Weeks 0, 4, and every 8 weeks through Week 24.Starting at Week 24, all subjects were treated with IV golimumab throughWeek 100. It was demonstrated that IV golimumab provided substantialbenefits in improving RA signs and symptoms, physical function, andhealth related quality of life, as well as inhibiting the progression ofstructural damage.

Golimumab administered intravenously in the treatment of RA(CNTO148ART3001) demonstrated robust efficacy and an acceptable safetyprofile with a low incidence of infusion reactions. This proposed Phase3 study is designed to evaluate the efficacy and safety of intravenous(IV) golimumab in the treatment of subjects with active AS. The IV routeof administration in subjects with AS is being evaluated since currentlyavailable IV anti-TNF α agents have limitations with respect toimmunogenicity and infusion reactions, and have longer infusion times(60 to 120 minutes) compared with the proposed 30±10 minute infusionswith IV golimumab.

Patients may also prefer the maintenance dosage schedule of q8w IVgolimumab rather than more frequent administrations compared with SCagents. Therefore, IV golimumab may be an important addition to thecurrently available treatment options.

The dosing regimen for this study is 2 mg/kg of golimumab administeredvia IV infusion over 30 minutes at Weeks 0 and 4, then q8w.

Study Design and Rationale

Overview of Study Design

This is a Phase 3 multicenter, randomized, double-blind,placebo-controlled study of the efficacy and safety of IV golimumabcompared with placebo in subjects with active AS with an inadequateresponse or intolerance to NSAIDs. Approximately 200 subjects will berandomized at approximately 70 investigational sites. Subjects will berandomly assigned to receive golimumab 2 mg/kg or placebo IV infusionsat Weeks 0, 4, and 12. At Week 16, all subjects receiving placeboinfusions will cross over and begin receiving golimumab IV infusions atWeeks 16, 20, and q8w thereafter through Week 52. Subjects in thegolimumab IV treatment group will receive a placebo infusion at Week 16to maintain the blind and continue to receive golimumab IV infusions atWeek 20 and q8w thereafter through Week 52. Database locks (DBL) arescheduled for Weeks 28 and 60.

Subjects will be followed for adverse events (AE) and serious adverseevents (SAE) at least 8 weeks following the last study treatmentadministration. The end of study is defined as the time the last subjectcompletes the Week 60 visit.

A diagram of the study design is provided in FIG. 18.

Study Design Rationale

Study Population

The target study population is subjects with active AS, as defined bythe modified New York criteria, for at least 3 months prior to firstadministration of study agent.

Treatment Groups, Dosage, and Dose Administrations Interval

Subjects will be randomized at Week 0 to 1 of 2 treatment groups asfollows:

Group 1 (n=100): IV placebo infusions

Group 2 (n=100): IV golimumab 2 mg/kg infusions

Subjects randomly assigned to golimumab will receive golimumab 2 mg/kgIV infusions at Weeks 0, 4, and q8w thereafter through Week 52. At Week16, subjects randomized to golimumab will receive a placebo infusion tomaintain the blind. All subjects randomly assigned to receive placebo IVinfusions at Weeks 0, 4, and 12 will crossover to active treatment atWeek 16 and receive golimumab 2 mg/kg IV infusions at Weeks 16, 20, andq8w thereafter through Week 52. Subjects in the golimumab IV treatmentgroup will continue to receive golimumab IV infusions.

Study Phases and Duration of Treatment

There will be 4 phases in this study: Screening, double-blindplacebo-controlled, active treatment, and safety follow-up. Thescreening phase of up to 6 weeks will allow for sufficient time toperform screening study evaluations and determine study eligibility. Thesecond phase of the study will be the double blind, placebo-controlledphase from Week 0 to Week 16. The third phase of the study will be theactive treatment phase from Week 16 through Week 52. The fourth phase ofthe study will be the safety follow-up phase and will be 8 weeks fromthe last administration of study agent. The safety follow-up allows formonitoring of the subject for a period equivalent to approximately 5times the half-life of golimumab. Initial treatment assignment for eachsubject is still blinded to sites and subjects throughout the 60 weeksof the study. This duration will provide adequate time to demonstratethe efficacy and safety of IV golimumab as maintenance therapy for AS.

The study will end when the last subject completes the last scheduledvisit (Week 60 visit).

Study Control, Randomization, and Blinding

Randomization will be used to minimize bias in the assignment ofsubjects to treatment groups, to increase the likelihood that known andunknown subject attributes (egg, demographic and baselinecharacteristics) are evenly balanced across treatment groups, and toenhance the validity of statistical comparisons across treatment groups.In addition, randomization will be stratified based on geographic regionand prior use of anti-TNFα therapy (yes or no).

Individual subjects and investigators will remain blinded for theduration of the study. Blinded treatment will be used to reducepotential bias during data collection and evaluation of clinicalendpoints. Two DBLs are planned for the study at Week 28 and Week 60.The first DBL will occur after all subjects complete the Week 28 visitor terminated study participation. The second DBL will occur after allsubjects have either completed the Week 60 visit or terminated studyparticipation. The database will be locked at Week 28 and summary-leveldata will be unblinded to subject-level data at DBL for data analysesand data review for selected individuals. All site personnel andsubjects will remain blinded to the treatment assignments with theexception of the unblinded pharmacist, until the Week 60 DBL hasoccurred.

Efficacy Evaluations

Efficacy evaluations chosen for this study were established in previoustrials of therapeutic biologic agents for the treatment of AS. Patientreported outcomes (PROs) chosen for this study are also consistent withclinically relevant measurements that are accepted in the medicalliterature for other studies in AS and applicable US/EU regulatoryguidance documents.

Ankylosing spondylitis response evaluations include:

Bath Ankylosing Spondylitis Functional Index (BASFI)

Patient's Global Assessment

Total Back Pain

Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)

36-item short form health survey (SF-36)

Bath Ankylosing Spondylitis Metrology Index (BASMI)

Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire

Chest Expansion

Night Back Pain

Enthesitis Index

Medical Outcomes Study Sleep scale

Work Limitations Questionnaire (WLQ)

Productivity Visual Analog Scale

EuroQol-5D (EQ-5D) Questionnaire

Subject Population

Subjects eligible for the study will be men or women 18 years of age orolder with a diagnosis of AS for at least 3 months defined as “definite”by the modified New York criteria, and symptoms of active disease, asevidenced by BASDAI ≥4, and a visual analogue scale (VAS) for total backpain of ≥4, each on a scale of 0 to 10 cm. Subjects are required to havea C-reactive protein (CRP) level of ≥0.3 mg/dL.

Other major features of the study population are as follows:

-   -   Current users of MTX, SSZ, and hydroxychloroquine (HCQ) and low        dose oral corticosteroids are permitted and should enter the        study on stable doses of these medications.    -   Subjects with prior exposure to no more than one biologic        anti-TNFα agent (other than golimumab) are permitted to be        included in the study, but will be limited to at most 20% of the        study population.    -   Subjects with complete ankylosis of the spine, defined as        bridging syndesmophytes present at all intervertebral levels of        the cervical and lumbar spine visualized on lateral-view spinal        radiographs are permitted to be included in the study, but will        be limited to at most 10% of the study population.

Screening for eligible subjects will be performed within 6 weeks beforeadministration of the study agent.

The inclusion and exclusion criteria for enrolling subjects in thisstudy are described in the following 2 subsections. If there is aquestion about the inclusion or exclusion criteria below, theinvestigator should consult with the appropriate representative beforeenrolling a subject in the study.

Inclusion Criteria

Each potential subject must satisfy all of the following criteria to beenrolled in the study.

1. Subject must be a man or woman 18 years of age or older.

2. Subject must be medically stable on the basis of physicalexamination, medical history, vital signs, and 12-lead electrocardiogram(ECG) performed at screening. This determination must be recorded in thesubject's source documents and initialed by the investigator.

3. Subject must be medically stable on the basis of clinical laboratorytests performed at screening. If the results of the serum chemistrypanel including liver enzymes or hematology are outside the normalreference ranges, the subject may be included only if the investigatorjudges the abnormalities or deviations from normal to be not clinicallysignificant or to be appropriate and reasonable for the population understudy. This determination must be recorded in the subject's sourcedocuments and initialed by the investigator. For tests described ininclusion criteria #6 and #16, results MUST be within the eligibilityranges allowed in inclusion criteria #6 and #16.

4. Have a diagnosis of definite AS, as defined by the modified New Yorkcriteria, for at least 3 months prior to first administration of studyagent.

-   -   Both the radiographic criterion and at least 1 clinical        criterion must be met:    -   a. Radiographic criterion: Sacroiliitis Grade ≥2 bilaterally or        sacroiliitis Grade 3 to 4 unilaterally.    -   b. Clinical criteria (at least 1):        -   i. Low back pain and stiffness for more than 3 months, which            improves with exercise, but is not relieved by rest.        -   ii. Limitation of motion of the lumbar spine in both the            sagittal and frontal planes.        -   iii. Limitation of chest expansion relative to normal values            corrected for age and sex.

5. Have symptoms of active disease at screening and at baseline, asevidenced by both a BASDAI score of ≥4 and a VAS score for total backpain of each on a scale of 0 to 10 cm.

6. Have a CRP level of ≥0.3 mg/dL at screening.

7. Either has an inadequate response to at least 2 NSAIDs over a 4 weekperiod in total with maximal recommended doses of NSAID(s), or is unableto receive a full 4 weeks of maximal NSAID therapy because ofintolerance, toxicity, or contraindications to NSAIDs.

8. If using NSAIDs or other analgesics for AS, must be on a stable dosefor at least 2 weeks prior to the first administration of study agent.If currently not using NSAIDs or other analgesics for AS, must not havereceived NSAIDs or other analgesics for AS for at least 2 weeks prior tothe first administration of the study agent.

9. If using oral corticosteroids, must be on a stable dose equivalent to≥10 mg of prednisone/day for at least 2 weeks prior to the firstadministration of study agent. If currently not using corticosteroids,must have not received oral corticosteroids for at least 2 weeks priorto the first administration of the study agent.

10. If using MTX, SSZ, or HCQ, should have started treatment at least 3months prior to the first administration of study agent and should haveno serious toxic side effects attributable to the disease modifyingantirheumatic drug (DMARD). Methotrexate routes of administration anddoses (not to exceed 25 mg/week) should be stable for at least 4 weeksprior to the first administration of the study agent. If using SSZ orHCQ, must also be on a stable dose for at least 4 weeks prior to thefirst administration of study agent. If currently not using MTX, SSZ, orHCQ, must have not received these DMARDs for at least 4 weeks prior tothe first administration of the study agent.

11. Before randomization, a woman must be either

-   -   Not of childbearing potential: premenarchal; postmenopausal (>45        years of age with amenorrhea for at least 12 months);        permanently sterilized (egg, tubal occlusion, hysterectomy,        bilateral salpingectomy); or otherwise be incapable of        pregnancy,    -   Of childbearing potential and practicing a highly effective        method of birth control consistent with local regulations        regarding the use of birth control methods for subjects        participating in clinical studies: egg, established use of oral,        injected or implanted hormonal methods of contraception;        placement of an intrauterine device or intrauterine system;        barrier methods: Condom with spermicidal        foam/gel/film/cream/suppository or occlusive cap (diaphragm or        cervical/vault caps) with spermicidal        foam/gel/film/cream/suppository; male partner sterilization (the        vasectomized partner should be the sole partner for that        subject); true abstinence (when this is in line with the        preferred and usual lifestyle of the subject).

12. A woman of childbearing potential must have a negative serumpregnancy test (β-human chorionic gonadotropin [β-HCG-1) at screeningand a negative urine pregnancy test on Week 0 before randomization.

13. A woman must agree not to become pregnant or to donate eggs (ova,oocytes) for the purposes of assisted reproduction during the study andfor 4 months after receiving the last dose of study agent.

14. A man who is sexually active with a woman of childbearing potentialand has not had a vasectomy must agree to use a barrier method of birthcontrol egg, either condom with spermicidalfoam/gel/film/cream/suppository or partner with occlusive cap (diaphragmor cervical/vault caps) with spermicidal foam/gel/film/cream/suppositoryduring the study and for 4 months after the last dose of study agent.All men must also not donate sperm during the study and for 4 monthsafter receiving the last dose of study agent.

15. Are considered eligible according to the following tuberculosis (TB)screening criteria:

-   -   a. Have no history of latent or active TB prior to screening. An        exception is made for subjects who have a history of latent TB        and are currently receiving treatment for latent TB, will        initiate treatment for latent TB prior to first administration        of study agent, or have documentation of having completed        appropriate treatment for latent TB within 5 years prior to the        first administration of study agent.    -   b. Have no signs or symptoms suggestive of active TB upon        medical history and/or physical examination.    -   c. Have had no recent close contact with a person with active TB        or, if there has been such contact, will be referred to a        physician specializing in TB to undergo additional evaluation        and, if warranted, receive appropriate treatment for latent TB        prior to the first administration of study agent.    -   d. Within 6 weeks prior to the first administration of study        agent, have a negative QuantiFERON®-TB Gold test result, or have        a newly identified positive QuantiFERON®-TB Gold test result in        which active TB has been ruled out and for which appropriate        treatment for latent TB has been initiated prior to the first        administration of study agent. Within 6 weeks prior to the first        administration of study agent, a negative tuberculin skin test        (TST), or a newly identified positive TST in which active TB has        been ruled out and for which appropriate treatment for latent TB        has been initiated prior to the first administration of study        agent, is additionally required if the QuantiFERON®-TB Gold test        is not approved/registered in that country or the TST is        mandated by local health authorities.        -   i. Subjects with persistently indeterminate QuantiFERON®-TB            Gold test results may be enrolled without treatment for            latent TB, if active TB is ruled out, their chest radiograph            shows no abnormality suggestive of TB (active or old,            inactive TB), and the subject has no additional risk factors            for TB as determined by the investigator.        -   ii. The QuantiFERON®-TB Gold test and the TST is/are not            required at screening for subjects with a history of latent            TB and ongoing treatment for latent TB or documentation of            having completed adequate treatment as described above;            Subjects with documentation of having completed adequate            treatment as described above are not required to initiate            additional treatment for latent TB.    -   e. Have a chest radiograph (posterior-anterior view) taken        within 3 months prior to the first administration of study agent        and read by a qualified radiologist, with no evidence of        current, active TB or old, inactive TB.

16. Have screening laboratory test results within the followingparameters:

-   -   a. Hemoglobin ≥8.5 g/dL    -   b. White blood cells ≥3.5×103/μL    -   c. Neutrophils ≥1.5×103/μL    -   d. Platelets ≥100×103/μL    -   e. Serum creatinine ≥1.5 mg/dL    -   f AST, ALT, and alkaline phosphatase levels must be within 1.5        times the ULN range for the laboratory conducting the test.

17. Subject must be willing and able to adhere to the prohibitions andrestrictions specified in this protocol.

18. Each subject must sign an informed consent form (ICF) indicatingthat he or she understands the purpose of and procedures required forthe study and are willing to participate in the study.

19. Each subject must sign a separate informed consent form if he or sheagrees to provide an optional DNA sample for research (where localregulations permit). Refusal to give consent for the optional DNAresearch sample does not exclude a subject from participation in thestudy.

20. Are willing to refrain from the use of complementary therapiesincluding ayurvedic medicine, traditional Chinese medication(s), andacupuncture within 2 weeks prior to the first study agent administrationand throughout the duration of the study.

Exclusion Criteria

Any potential subject who meets any of the following criteria will beexcluded from participating in the study.

1. Have other inflammatory diseases that might confound the evaluationsof benefit from the golimumab therapy, including but not limited to, RA,PsA, systemic lupus erythematosus, or Lyme disease.

2. Are pregnant, nursing, or planning a pregnancy or fathering a childwhile enrolled in the study or within 4 months after receiving the lastadministration of study agent.

3. Have received any systemic immunosuppressives or DMARDs other thanMTX, SSZ, or HCQ within 4 weeks prior to first administration of studyagent. Medications in these categories include, but are not limited tochloroquine, azathioprine, cyclosporine, mycophenolate mofetil, gold,and penicillamine. Corticosteroids are not included in this criterion;see other eligibility criteria regarding corticosteroids.

4. Have received leflunomide within 4 weeks prior to the firstadministration of study agent (irrespective of undergoing a drugelimination procedure), or have received leflunomide within 3 monthsprior to the first administration of study agent and have not undergonea drug elimination procedure.

5. Have received epidural, intra-articular, IM, or IV corticosteroids,including adrenocorticotropic hormone during the 4 weeks prior to firstadministration of study agent.

6. Have ever received golimumab.

7. Have received infliximab (including biosimilar anti-TNFα agents),adalimumab, or certolizumab pegol within 3 months prior to the firstadministration of the study agent.

8. Have received etanercept or yisaipu within 6 weeks prior to the firstadministration of the study agent.

9. Have received more than one prior anti-TNFα agent.

10. Have experienced primary failure to any prior anti-TNFα agent(defined as lack of response as assessed by the investigator ordiscontinuation due to lack of efficacy within the first 16 weeks oftreatment).

11. Have received prior biologic therapy other than anti-TNFα agents,including but not limited to tocilizumab, alefacept, efalizumab,natalizumab, abatacept, anakinra, ustekinumab, brodalumab, secukinumab,ixekizumab, and B-cell depleting therapies.

12. Have ever received tofacitinib or any other Janus kinase inhibitors(JAK) inhibitor

13. Have a known hypersensitivity to human immunoglobulin proteins.

14. Have used cytotoxic drugs, including chlorambucil, cyclophosphamide,nitrogen mustard, or other alkylating agents.

15. Have a history of active granulomatous infection, includinghistoplasmosis, or coccidioidomycosis, prior to screening. Refer toinclusion criteria for information regarding eligibility with a historyof latent TB.

16. Have had a Bacille Calmette-Guérin (BCG) vaccination within 12months of screening.

17. Have a chest radiograph within 3 months prior to the firstadministration of study agent that shows an abnormality suggestive of amalignancy or current active infection, including TB.

18. Have had a nontuberculous mycobacterial infection or opportunisticinfection (egg, cytomegalovirus, pneumocystosis, aspergillosis) within 6months prior to screening.

19. Have had a herpes zoster infection within 2 months of firstadministration of study agent.

20. Have received, or are expected to receive, any live virus orbacterial vaccination within 3 months before the first administration ofstudy agent, during the study, or within 3 months after the lastadministration of study agent.

21. Have a history of an infected joint prosthesis, or have receivedantibiotics for a suspected infection of a joint prosthesis, if thatprosthesis has not been removed or replaced.

22. Have had a serious infection (including but not limited to,hepatitis, pneumonia, sepsis, or pyelonephritis), or have beenhospitalized for an infection, or have been treated with IV antibioticsfor an infection within 2 months prior to first administration of studyagent.

23. Have a history of, or ongoing, chronic or recurrent infectiousdisease, including but not limited to, chronic renal infection, chronicchest infection (egg, bronchiectasis), sinusitis, recurrent urinarytract infection (egg, recurrent pyelonephritis), an open, draining, orinfected skin wound, or an ulcer.

24. Subject has a history of human immunodeficiency virus (HIV) antibodypositive, or tests positive for HIV at Screening.

25. Has a hepatitis B infection. Subjects must undergo screening forhepatitis B virus (HBV). At a minimum, this includes testing for HBsAg(HBV surface antigen), anti-HBs (HBV surface antibody), and anti-HBctotal (HBV core antibody total).

26. Subjects who are seropositive for antibodies to hepatitis C virus(HCV), unless they have 2 negative HCV RNA test results 6 months apartprior to screening and have a third negative HCV RNA test result atscreening.

27. Have a history of known demyelinating diseases such as multiplesclerosis or optic neuritis.

28. Have current signs or symptoms of severe, progressive, oruncontrolled renal, hepatic, hematological, gastrointestinal, endocrine,pulmonary, cardiac, neurologic, cerebral, or psychiatric disease.

29. Have a history of, or concurrent congestive heart failure (CHF),including medically controlled, asymptomatic CHF.

30. Have a known history of lymphoproliferative disease, includinglymphoma, or signs and symptoms suggestive of possiblelymphoproliferative disease, such as lymphadenopathy of unusual size orlocation, clinically significant splenomegaly, or monoclonal gammopathyof undetermined significance.

31. Subject has a history of malignancy within 5 years before screening(exceptions are squamous and basal cell carcinomas of the skin that hasbeen treated with no evidence of recurrence for at least 3 months beforethe first study agent administration and carcinoma in situ of the cervixthat has been surgically cured).

32. Subject has known allergies, hypersensitivity, or intolerance togolimumab or its excipients (refer to the golimumab IB).

33. Subject has taken any disallowed therapies, before the planned firstdose of study drug.

34. Subject has received an investigational drug (includinginvestigational vaccines) within 5 half-lives or 3 months, whichever islonger, or used an invasive investigational medical device within 3months before the planned first dose of study drug or is currentlyenrolled in an investigational study.

35. Subject has any condition for which, in the opinion of theinvestigator, participation would not be in the best interest of thesubject (egg, compromise the well-being) or that could prevent, limit,or confound the protocol-specified assessments.

36. Subject has had major surgery, (egg, requiring general anesthesia)within 1 month before screening, or will not have fully recovered fromsurgery, or has surgery planned during the time the subject is expectedto participate in the study or within 1 month after the last dose ofstudy drug administration.

37. Have a transplanted organ (with the exception of a cornealtransplant performed >3 months prior to first administration of studyagent).

38. Have or have had a substance abuse (drug or alcohol) problem withinthe previous 3 years.

39. Are unwilling or unable to undergo multiple venipunctures because ofpoor tolerability or lack of easy access.

40. Subject is an employee of the investigator or study site, withdirect involvement in the proposed study or other studies under thedirection of that investigator or study site, as well as family membersof the employees or the investigator.

NOTE: Investigators should ensure that all study enrollment criteriahave been met at screening and again prior to randomization. If asubject's status changes (including laboratory results or receipt ofadditional medical records) after screening but before the first dose ofstudy drug is given such that he or she no longer meets all eligibilitycriteria, then the subject should be excluded from participation in thestudy.

Prohibitions and Restrictions

Potential subjects must be willing and able to adhere to the followingprohibitions and restrictions during the course of the study to beeligible for participation:

1. Both heterosexually active women of childbearing potential and mencapable of fathering a child must consent to use a highly effectivemethod of contraception and continue to use contraception for theduration of the study and for 4 months after the last administration ofstudy agent.

2. The use of the following drugs is not permitted concomitantly with IVstudy agent administration:

-   -   Systemic immunosuppressives or DMARDs (other than MTX, SSZ, and        HCQ) including chloroquine, azathioprine, oral cyclosporine A,        tacrolimus, mycophenolate mofetil, leflunomide, oral or        parenteral gold. Systemic immunosuppressives do not refer to        corticosteroids; see elsewhere regarding corticosteroid        restrictions.    -   Biologic agents targeted at reducing TNFα (including but not        limited to infliximab, SC golimumab, certolizumab pegol,        etanercept, yisaipu, CT-P13 [Remsima®1 and adalimumab)    -   IL-lra (anakinra)    -   Tocilizumab or any other biologic targeting IL-6 or IL-6        receptor    -   Tofacitinib or any other JAK inhibitor    -   B-cell depleting agents (egg, rituximab)    -   Cytotoxic drugs such as cyclophosphamide, chlorambucil, nitrogen        mustard, or other alkylating agents    -   Abatacept    -   Ustekinumab    -   Anti-IL-17 agents (egg, brodalumab, secukinumab, and ixekizumab)    -   Investigational drugs

3. Must agree not to receive a live virus or live bacterial vaccinationduring the study. Subjects must also agree not to receive a live vaccinefor 3 months after receiving the last administration of study agent.Must not have had a BCG vaccination within 12 months of screening.

4. Must agree not to receive an investigational medical device or aninvestigational drug other than study agent for the duration of thisstudy.

5. Subjects treated with NSAIDs, including aspirin and selective COX-2inhibitors, and other analgesics should receive the usual marketed dosesapproved in the country in which the study is being conducted.Prescriptions of NSAIDs and other analgesics should not be adjusted forat least 2 weeks prior to the first administration of the study drug,and through Week 16, and may be changed only if the subject developsunacceptable side effects. After Week 16 through Week 60, a one-timedose decrease is allowed; otherwise, prescriptions of NSAIDs and otheranalgesics may be changed only if the subject develops unacceptable sideeffects.

The use of topical analgesics including capsaicin and diclofenac isallowed.

6. Subjects treated with oral corticosteroids should receive a stabledose equivalent to ≤10 mg prednisone per day for at least 2 weeks priorto their first administration of the study agent and continue to receivethis dose through Week 16. After Week 16 and through Week 60, a one-timedose decrease in oral corticosteroids is allowed; otherwise the dose andtype of oral corticosteroid may be changed at the discretion of theinvestigator only if the subject develops unacceptable side effects.

Epidural, IM or IV administration of corticosteroids is not allowedwithin 4 weeks before the first administration of study agent and is notallowed for the treatment of AS throughout the study. Every attemptshould be made to avoid the use of epidural, IM, and IV corticosteroidsduring the study for indications other than AS. Long-term (>2 weeks)oral or IV corticosteroids use for indications other than AS are notallowed through Week 60. Short-term weeks) oral, IV, IM, or epiduralcorticosteroid used for indications other than AS should be limited tosituations where, in the opinion of the treating physician, there are noadequate alternatives.

Intra-articular steroids should not be administered within 4 weeks priorto the first administration of study agent. Attempts should be made toavoid intra-articular corticosteroid injections especially during thefirst 16 weeks of the study. However if necessary, subjects may receiveup to 2 intra-articular, tendon sheath, or bursal corticosteroidinjections in no more than 2 affected sites during the 60 weeks of thestudy.

7. The use of complementary therapies that may affect AS diseaseactivity or assessments, including but not limited to traditionalmedicine (egg, Chinese, acupuncture, ayurvedic medicine) is prohibitedthrough Week 60.

Prestudy and Concomitant Therapy

Every effort should be made to keep subjects' concomitant medicationsstable through Week 16 or as specified for AS therapies in the followingsections. The concomitant medication dose may be reduced or themedication temporarily discontinued because of abnormal laboratoryvalues, side effects, concurrent illness, or the performance of asurgical procedure, but the change and reason for the change should beclearly documented in the subject's medical record.

Subjects should not initiate any new treatment for AS during the 60-weekstudy period.

Concomitant medication review will occur at study visits identified inthe Time and Events Schedule.

Methotrexate, Sulfasalazine, or Hydroxychloroquine

Subjects are permitted to enter the study on stable doses of MTX, SSZ,or HCQ.

If subjects are using MTX, SSZ, or HCQ, treatment should have started atleast 3 months prior to the first administration of study agent. MTXroutes of administration and doses ≤25 mg/week should be stable for atleast 4 weeks prior to the first administration of the study agent. Itis recommended that all subjects taking MTX in this study receive atleast 5 mg oral folate or 5 mg folinic acid weekly. If using SSZ or HCQ,subjects must also be on a stable dose for at least 4 weeks prior to thefirst administration of study agent.

Subjects not on treatment with MTX, SSZ, or HCQ must have discontinuedthe treatment for at least 4 weeks prior to the first administration ofstudy agent, and must not receive MTX, SSZ, or HCQ through Week 60.

Every effort should be made to maintain stable doses and routes ofadministration of MTX, SSZ, and HCQ through Week 60 of the study insubjects receiving this medication. Guidelines for dose adjustment inthe event of MTX toxicity are included in the Trial Center File.

Corticosteroid Therapy

Subjects treated with oral corticosteroids for AS should receive astable dose equivalent to =10 mg prednisone per day for at least 2 weeksprior to first administration of study agent and continue to receivethis dose through Week 16. After Week 16 and through Week 60, a one-timedose decrease in oral corticosteroids is allowed; otherwise the dose andtype of oral corticosteroid may be changed at the discretion of theinvestigator only if the subject develops unacceptable side effects.Subjects not treated with oral corticosteroids at baseline must havediscontinued oral corticosteroids at least 2 weeks prior to the firstadministration of study agent, and they must not receive oralcorticosteroids for AS through Week 60.

Intravenous, intramuscular, or epidural administration ofcorticosteroids for the treatment of AS is not allowed through Week 60.

Long-term (>2 weeks) oral or IV corticosteroids use for indicationsother than AS are not allowed through Week 60. Short-term (≤2 weeks)oral, IV, IM, or epidural corticosteroid used for indications other thanAS should be limited to situations where, in the opinion of the treatingphysician, there are no adequate alternatives. Inhaled, otic,ophthalmic, intranasal, and other routes of mucosal delivery ofcorticosteroids are allowed throughout the course of the study.

Attempts should be made to avoid intra-articular corticosteroidinjections especially during the first 16 weeks of the study. However ifnecessary, subjects may receive up to 2 intra-articular, tendon sheath,or bursal corticosteroid injections in no more than 2 affected sitesduring the 60 weeks of the study. In the case of severe tenderness orswelling in a single joint, it is suggested that the subject beevaluated for infection prior to receiving an intra-articularcorticosteroid injection.

Nonsteroidal Anti-inflammatory Drugs and Other Analgesics

The use of stable doses of NSAIDs and other analgesics is allowed.

Subjects treated with NSAIDs, including aspirin and selectivecyclooxygenase-2 inhibitors, and other analgesics should receive theusual marketed doses approved in the country in which the study is beingconducted, and should have been on a stable dose at least 2 weeks priorto the first administration of the study agent. Through Week 16, thedose and type of NSAIDs and other analgesics may be changed only if thesubject develops unacceptable side effects. After Week 16 and throughWeek 60, a one-time dose decrease is allowed; otherwise, prescriptionsof NSAIDs and other analgesics may be changed only if the subjectdevelops unacceptable side effects.

The use of topical analgesics including capsaicin and diclofenac isallowed.

In this trial, aspirin is considered an NSAID, except for low-doseaspirin prescribed for cardiovascular or cerebrovascular disease.

Disease Modifying Antirheumatic Drugs/Systemic Immunosuppressives

Disease modifying antirheumatic drugs/systemic immunosuppressive agents,with the exception of MTX, SSZ, and HCQ must be discontinued at least 4weeks prior to the first administration of study agent and areprohibited through Week 60. These DMARDs include, but are not limited tochloroquine, gold preparations, penicillamine, and leflunomide. If asubject received leflunomide within 3 months prior to the firstadministration of study agent, the subject must have undergone a drugelimination procedure. Prohibited systemic immunosuppressive drugsthrough Week 60, include, but are not limited to, cyclosporine,tacrolimus, mycophenolate mofetil, and azathioprine. Systemicimmunosuppressives do not refer to corticosteroids.

Biologic Agents, Cytotoxic Drugs, or Investigational Agents

The use of biologic agents (egg, SC golimumab, anakinra, etanercept,adalimumab, infliximab, alefacept, efalizumab, rituximab, natalizumab),cytotoxic agents (egg, chlorambucil, cyclophosphamide, nitrogen mustard,other alkylating agents), or investigational drugs is not allowed duringthe 60 weeks of the study. If any of these medications are used, thesubject will be discontinued from further study agent infusions.

Complementary Therapies

The use of complementary therapies including ayurvedic medicine,traditional Chinese medications or non-medicinal therapy such asacupuncture is not allowed during the 60 weeks of the study.

Study Evaluations

Efficacy Evaluations Bath Ankylosing Spondylitis Disease Activity Index

The BASDAI¹⁴ is defined below:

Subject self-assessment using a VAS (0 to 10 cm) on the followingcriteria:

A. Fatigue

B. Spinal pain

C. Joint pain

D. Enthesitis

E. Qualitative morning stiffness

F. Quantitative morning stiffness

The BASDAI=0.2 (A+B+C+D+0.5[E+F]).

Bath Ankylosing Spondylitis Functional Index

The BASFI is a subject's self-assessment represented as a mean (VAS; 0to 10 cm) of 10 questions, 8 of which relate to the subject's functionalanatomy and 2 of which relate to a subject's ability to cope witheveryday life.⁸ An increase along the scale indicates a worseningcondition.

Patient's Global Assessment

Patient's global assessment of disease activity will be recorded on aVAS (0 to 10 cm; 0=very well, 10=very poor).

Total Back Pain

Subjects will be asked to assess their average total back pain over thepast week on a VAS (0 to 10 cm; 0=no pain, 10=most severe pain).

Night Back Pain

Subjects will be asked to assess their nighttime back pain during thepast week on a VAS (0 to 10 cm; 0=no pain, 10=most severe pain).

Musculoskeletal Assessments

The musculoskeletal assessments will include each component of theBASMI, enthesitis index, and chest expansion.

An independent musculoskeletal assessor (IMA) with adequate training andexperience in performing musculoskeletal assessments will be designatedat each study site to perform all musculoskeletal assessments. It isstrongly recommended that the same IMA who performs the baselinemusculoskeletal assessments for a subject should also perform themusculoskeletal assessments for that subject at every subsequent visitthrough Week 52.

The Sponsor will provide training for each site's designated IMA priorto the screening of the first subject at each site. A back-up IMA mustcomplete training before performing a musculoskeletal assessment for asubject's study visit. Training documentation of each IMA should bemaintained at the study site. If possible, the independent assessor atthe study site should not be changed during the study.

If an IMA was trained by the Sponsor in a previous clinical study withinthe last 3 years and there is adequate documentation of this training(certification), that training will be considered adequate for thisstudy; however, repeat training prior to start of the trial isencouraged.

All IMAs performing the musculoskeletal evaluation at a site must belisted on the Delegation Log at the study site and should be documentedin the source documents at each visit.

After Week 28, the musculoskeletal assessor no longer needs to beindependent. However it is recommended that the musculoskeletal assessorshould not be changed during the study.

Bath Ankylosing Spondylitis Metrology Index

The BASMI is represented as an aggregate score of 5 components (rangingfrom 0 to 10) and will be calculated using the van der Heijdecalculation³¹ as shown in Table 6.

TABLE 6 Equations proposed for the conversion of the assessments (A)into scores (S) for the five components of the BASMI_(lin) S = 0 if:Between 0 and 10: S = 10 if: Lateral lumbar flexion* (cm) A ≥ 21.1 S =(21.1 − A)/2.1 A ≤ 0.1 Tragus-to-wall distance* (cm) A ≤ 8 S = (A − 8)/3A ≥ 38 Lumbar flexion (modified A ≥ 7.4 S = (7.4 − A)/0.7 A ≤ 0.4Schober) (cm) Intermalleolar distance (cm) A ≥ 124.5 S = (124.5 − A)/10A ≤ 24.5 Cervical rotation angle* (°) A ≥ 89.3 S = (89.3 − A)/8.5 A ≤4.3 *For lateral lumbar flexion, tragus-to-wall distance, and cervicalrotation the average of right and left should be taken. If a score liesbeyond the range 0-10, the values 0 or 10 have to be used, respectively.

The BASMI_(lin) is the mean of the five S scores.

The assessments (A) of the 5 components will be collected at the sitesand the scores (S) will be calculated programmatically based onassessments when analysis is performed.

Assessment in Ankylosing Spondylitis Response Criteria

A 20% improvement in response according to the criteria of the ASASInternational Working Group (ASAS 20)^(1,23,33) is defined as:

1. An improvement of ≥20% from baseline and absolute improvement frombaseline of at least 1 on a 0 to 10 cm scale in at least 3 of thefollowing 4 domains:

-   -   i. Patient global    -   ii. Total back pain    -   iii. Function (BASFI)    -   iv. Inflammation (average of the last 2 questions of the BASDAI        concerning morning stiffness)

2. Absence of deterioration from baseline (≥20% and worsening of atleast 1 on a 0 to 10 cm scale) in the potential remaining domain.

ASAS 40 is defined as a ≥40% improvement in 3 of 4 domains, with anabsolute improvement of at least 2 on a 0 to 10 cm scale, and nodeterioration in the remaining domain.

ASAS 5/6 is defined as a ≥20% improvement in any 5 of the 6 domains ofpain (VAS 0 to 10 cm), patient global (VAS 0 to 10 cm), function (BASFIscore), morning stiffness (from BASDAI), CRP, and spine mobility (lumbarside flexion).

Low Disease Activity

Low level of disease activity will be measured by criteria for “ASASpartial remission,” defined as a value below 2 on a scale of 0 to 10 cmin each of the 4 ASAS domains described above.

Disease Activity Score for Ankylosing Spondylitis

The Assessment of SpondyloArthritis international Society (ASAS) hasdeveloped a disease activity score (DAS) for use in AS, theASDAS.^(21, 32) For this study the following formula will be used tocalculate the ASDAS score:

ASDAS=0.121×Total back pain+0.058×Duration of morningstiffness+0.110×Patient global assessment+0.073×Peripheralpain/swelling+0.579×Ln (CRP (mg/L)+1).

Major improvement in ASDAS is defined as a decrease ≥2.0. Inactivedisease is defined as an ASDAS score <1.3.

Clinically important improvement in ASDAS is defined as a decrease ≥1.1²

Enthesitis Index

Enthesitis is an important feature of AS and otherspondyloarthropathies. The current University of California SanFrancisco (UCSF) enthesitis index used in clinical research will beperformed in this study.^(6,15) Evaluation of the listed entheses by theIMA will allow determination of the total enthesitis score (Table 7).

TABLE 7 Enthesitis Index Enthesis UCSF C1-2 X C7-T1 X T12-L1 X Ischialtuberosity L/R Symphysis pubis X 5th lumbar spinous process X Iliaccrest L/R Greater trochanter L/R Medial femoral condyle L/R Achillestendon insertion L/R Plantar insertion L/R TOTAL ENTHESES 17

Entheses are scored as either 0 (nontender) or 1 (tender).

Chest Expansion

Chest expansion is the difference, in cm, between the circumference ofthe chest in maximal inspiration and maximal expiration. It is measuredat the level of the fourth intercostal space in males, and just belowthe breasts in females.

36-Item Short-form Health Survey

The Medical Outcome Study health measure SF-36 questionnaire wasdeveloped as part of the Rand Health Insurance Experiment and consistsof 8 multi-item scales:

-   -   limitations in physical functioning due to health problems;    -   limitations in usual role activities due to physical health        problems;    -   bodily pain;    -   general mental health (psychological distress and well-being);    -   limitations in usual role activities due to personal or        emotional problems;    -   limitations in social functioning due to physical or mental        health problems;    -   vitality (energy and fatigue);    -   general health perception.

These scales are scored from 0 to 100 with higher scores indicatingbetter health. Another algorithm yields 2 summary scores, the PhysicalComponent Summary (PCS) and the Mental Component Summary (MCS). Thesesummary scores are also scaled with higher scores indicating betterhealth but are scored using a norm-based system where lineartransformations are performed to transform scores to a mean of 50 andstandard deviations of 10, based upon general US population norms.³⁵ Theconcepts measured by the SF-36 are not specific to any age, disease, ortreatment group, allowing comparison of relative burden of differentdiseases and the relative benefit of different treatments.³⁶

Medical Outcomes Study Sleep Scale

The extent of sleep problems will be assessed using the MOS-SS.¹⁷ MOS-SSmeasures six dimensions of sleep, including initiation, maintenance(egg, staying asleep), quantity, adequacy, somnolence (egg, drowsiness),and respiratory impairments (egg, shortness of breath, snoring). The MOSSleep Scale is a generic health measure, assessing a health-relatedquality of life (HRQOL) concept- sleep that is relevant to everyone'shealth status and wellbeing and known to be directly affected by diseaseand treatment. As such, the MOS-SS is not specific to any age, disease,or treatment group. The reliability and validity of the MOS-SS have beenevaluated in a number of disease areas, including neuropathic pain andRA.

Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire

Ankylosing spondylitis quality of life is a self-administeredpatient-reported outcomes instrument.¹² It consists of 18 itemsrequesting a Yes or No response to questions related to the impact ofpain on sleep, mood, motivation, ability to cope, activities of dailyliving, independence, relationships, and social life. A score of 1 isgiven to a response of ‘YES″ on each item and all item scores are summedto a total score with a range of 0-18. Higher scores indicate worsehealth related quality of life. Subjects can complete the instrument inless than four minutes.

EQ-5D Questionnaire

The EuroQo1-5D (EQ-5D) is a standardized measure of health statusdeveloped by the EuroQoL Group to provide a simple, generic measure ofhealth for clinical and economic appraisal (EuroQoL Group, 1990).¹³ TheEQ-5D is applicable to a wide range of health conditions and treatments.EQ-5D essentially consists of 2 elements: The EQ-5D descriptive systemand the EQ visual analogue scale (EQ VAS). The EQ-5D descriptive systemcomprises the following 5 dimensions: mobility, self-care, usualactivities, pain/discomfort and anxiety/depression. Each dimension has 5levels: no problems, slight problems, moderate problems, severeproblems, and extreme problems. The respondent is asked to indicatehis/her health state by ticking (or placing a cross) in the box againstthe most appropriate statement in each of the 5 dimensions. Thisdecision results in a 1-digit number expressing the level selected forthat dimension. The digits for 5 dimensions can be combined in a 5-digitnumber describing the respondent's health state which can be convertedinto a single summary index (EQ-5D index) by applying a formula thatattaches values (also called weights) to each of the levels in eachdimension. The EQ VAS records the respondent's self-rated health on avertical line, VAS where the endpoints are labeled ‘Best imaginablehealth state’ and ‘Worst imaginable health state’. The EQ VAS can beused as a quantitative measure of health outcome as judged by theindividual respondents.

Endpoints

Primary Endpoint

The primary endpoint of this study is the proportion of subjects whoachieve an ASAS 20 response at Week 16.

The study will be considered positive if the proportion of subjects withASAS 20 at Week 16 is demonstrated to be statistically significantlygreater in the golimumab group compared with the placebo group.

Major Secondary Endpoints

The following major secondary endpoints are listed in order ofimportance as specified below:

1. The proportion of subjects who achieve an ASAS 40 response at Week16.

2. The proportion of subjects who achieve at least a 50% improvementfrom baseline in BASDAI at Week 16.

3. The change from baseline in BASFI at Week 16.

Other Secondary Endpoints

Controlled secondary endpoints (with control of Type I error rate formultiplicity).

The following controlled secondary endpoints will be analyzed inaddition to the primary and major secondary endpoints and are listed inthe order of importance as specified below:

1. The change from baseline in SF-36 PCS at Week 16.

2. The change from baseline in SF-36 MCS at Week 16.

3. The proportion of subjects who achieve low level of disease activity(ASAS partial remission) at Week 16.

4. The change from baseline in ASQoL at Week 16.

5. The change from baseline in BASMI at Week 16.

To control for multiplicity, the above endpoints will be testedsequentially according to the above order only when the primary and allmajor secondary endpoints achieve statistical significance.

Other Secondary Endpoints Include

In addition to the primary, major secondary, and controlled secondaryendpoints, the following endpoints will be evaluated:

1. The proportion of subjects who achieve an ASAS 20 response at Week 2.

2. The proportion of subject who achieve an ASAS 20 response and an ASAS40 response over time.

3. The proportion of subjects who achieve low disease activity (ASASpartial remission) over time.

4. The change in baseline in BASFI over time.

5. The change from baseline in BASMI over time.

6. The change from baseline in the PCS and MCS scores of SF-36 overtime.

7. The proportion of subjects who achieve a BASDAI score of <3 overtime.

8. The change from baseline in ASDAS over time.

9. The proportion of subjects who achieve ASDAS major improvement(decrease ≥2.0) over time.

10. The proportion of subjects who achieve ASDAS inactive disease (<1.3)over time.

11. The change from baseline in the enthesitis scores in subjects withenthesitis at baseline over time.

12. The change from baseline in ASQoL scores over time.

Subject Completion/Withdrawal

Completion

A subject will be considered to have completed the study if he or shehas completed assessments at Week 60 of the study. Subjects whoprematurely discontinue study treatment for any reason will not beconsidered to have completed the study.

Withdrawal from the Study

A subject will be withdrawn from the study for any of the followingreasons:

-   -   Lost to follow-up    -   Withdrawal of consent    -   Death

If a subject is lost to follow-up, every reasonable effort must be madeby the study site personnel to contact the subject and determine thereason for discontinuation/withdrawal. The measures taken to follow upmust be documented.

When a subject withdraws before completing the study, the reason forwithdrawal is to be documented in the eCRF and in the source document.Study drug assigned to the withdrawn subject may not be assigned toanother subject. Subjects who withdraw will not be replaced. If asubject discontinues from the study agent administrations before the endof treatment, posttreatment assessments should be obtained.

Withdrawal of Participation in the Collection of Optional ResearchSamples While Remaining in the Main Study

The subject may withdraw consent for optional research samples whileremaining in the study. In such a case, the optional research sampleswill be destroyed. The sample destruction process will proceed asdescribed above.

Withdrawal from the Use of Samples in Future Research

The subject may withdraw consent for use of samples for research. Insuch a case, samples will be destroyed after they are no longer neededfor the clinical study. Details of the sample retention for research arepresented in the main ICF and in the separate ICF for optional researchsamples.

Statistical Methods

Simple descriptive summary statistics, such as n, mean, SD, median, IQrange, minimum, and maximum for continuous variables, and counts andpercentages for discrete variables will be used to summarize most data.

The Cochran-Mantel-Haenszel (CMH) test stratified by prior use ofanti-TNFα therapy will be used to compare categorical variables such asthe proportion of subjects responding to treatment, unless otherwisestated. In general, ANOVA with prior use of anti-TNFα therapy as afactor will be used for analyzing continuous variables, unless otherwisestated. All statistical tests will be performed at α=0.05 (2-sided). Inaddition to statistical analyses, graphical data displays (egg, lineplots) and subject listings may also be used to summarize/present thedata.

Efficacy analyses and summaries of subject information will be based onthe intent-to-treat population (i.e., all randomized subjects). Subjectsincluded in the efficacy analyses will be summarized according to theirassigned treatment group regardless of whether or not they receive theassigned treatment.

Safety and PK analyses will include all subjects who received at leastone administration of study treatment.

Efficacy Analyses

Primary Endpoint Analysis

The primary endpoint of this study is the proportion of subjects whoachieve an ASAS 20 response at Week 16.

To address the primary objective, the proportion of subjects who achievean ASAS 20 response at Week 16 will be compared between the placebo andgolimumab groups using a CMH test stratified by prior use of anti-TNFαtherapy (yes or no) at a significance level of 0.05 (2-sided).

In this primary efficacy analysis, data from all randomized subjectswill be analyzed according to their assigned treatment group regardlessof their actual treatment received. A last observation carried forward(LOCF) procedure will be used to impute the missing ASAS components ifthe subjects have data for at least 1 ASAS component at Week 16. If thesubjects do not have data for all the ASAS components at Week 16, thesubjects will be considered non-responders. In addition, treatmentfailure rules will be applied.

In addition, subgroup analysis will be performed to evaluate consistencyin the primary efficacy endpoint by demographic characteristics,baseline disease characteristics, and baseline medications. Interactiontest between the subgroups and treatment group will also be provided ifappropriate.

Major Secondary Analyses

The following major secondary analyses will be performed in order ofimportance as specified below:

1. The proportion of subjects who achieve an ASAS 40 at Week 16 will besummarized and compared between treatment groups.

2. The proportion of subjects who achieve at least a 50% improvementfrom baseline in

BASDAI at Week 16 will be summarized and compared between treatmentgroups.

3. The change from baseline in BASFI at Week 16 will be summarized andcompared between treatment groups.

Since there are only 2 treatment groups (1 statistical comparison),there is no need to adjust for multiplicity within each efficacyendpoint.

To control the Type I error rate for multiplicity, the first majorsecondary endpoint will be tested only if the primary endpoint achievedstatistical significance at a 0.05 level of significance (2-sided). Thesubsequent major secondary endpoints will be tested only if the primaryendpoint and the preceding major secondary endpoint(s) are statisticallysignificant at a 0.05 level of significance (2-sided).

Other Planned Efficacy Analyses

Controlled Secondary Endpoints (with Control of Type I Error Rate forMultiplicity).

The following efficacy analyses will be performed in addition to theprimary and major secondary analyses:

1. The change from baseline in SF-36 PCS at Week 16 will be summarizedand compared between treatment groups.

2. The change from baseline in SF-36 MCS at Week 16 will be summarizedand compared between treatment groups.

3. The proportion of subjects who achieve low level of disease activity(ASAS partial remission) at Week 16 will be summarized and comparedbetween treatment groups.

4. The change from baseline in ASQoL at Week 16 will be summarized andcompared between treatment groups.

5. The change from baseline in BASMI at Week 16 will be summarized andcompared between treatment groups.

To control for multiplicity, the above analyses, will be performedsequentially according to the above order only when all primary andmajor secondary endpoints achieved statistical significance. Otherwise,nominal p-values will be provided.

Other Secondary Endpoints Include

The following endpoints will be summarized by treatment groups.Summaries will be over time through Week 52 if the visit of the endpointis not specified. Comparisons between treatment groups will be made atvisits prior to and at Week 16.

1. The proportion of subjects who achieve an ASAS 20 response at Week 2will be summarized by treatment group and compared between groups

2. The proportion of subjects who achieve an ASAS 20 response and anASAS 40 response.

3. The proportion of subjects who achieve low disease activity (ASASpartial remission).

4. The change in baseline in BASFI.

5. The change from baseline in BASMI.

6. The change from baseline in the PCS and MCS scores of SF-36.

7. The proportion of subjects who achieve a BASDAI score of <3.

8. The change from baseline in ASDAS.

9. The proportion of subjects who achieve ASDAS major improvement(decrease >2.0).

10. The proportion of subjects who achieve ASDAS inactive disease(<1.3).

11. The change from baseline in the enthesitis scores in subjects withenthesitis at baseline.

12. The change from baseline in ASQoL scores.

Criteria for Endpoints

The study will be considered positive if the proportion of subjects withASAS 20 at Week 16 is demonstrated to be statistically significantlygreater in the golimumab group compared with the placebo group.

Study Drug Information

Physical Description of Study Drug Golimumab

The 50 mg Golimumab Final Vialed Product (FVP) for IV administration issupplied as a single use, sterile solution containing CNTO 148 IgG in a4 mL, Type I glass vial. Each vial contains 4 mL solution of 12.5 mg/mLgolimumab in an aqueous medium of histidine, sorbitol, and polysorbate80 at pH 5.5. No preservatives are present.

Placebo

Normal saline will be supplied as a sterile liquid for IV infusion insingle-use infusion bags. No preservatives are present.

Preparation, Handling, and Storage

At the study site, vials of golimumab solution must be stored in asecured refrigerator at 2° C. to 8° C. (35.6° F. to 46.4° F.), notfrozen and protected from light. Vigorous shaking of the product shouldbe avoided. Prior to administration, the product should be inspectedvisually for particulate matter and discoloration. If discoloration,visible particles, or other foreign particles are observed in thesolution, the product should not be used.

Study agent in glass vials will be ready for use. The study agent IVinfusions will be prepared according to the subject's weight by theunblinded pharmacist or other appropriately licensed and authorizedpersonnel. The pharmacist or other appropriately licensed and authorizedpersonnel will prepare the required volume of study agent usingappropriate number of vials.

Aseptic procedures must be used during the preparation andadministration of study material. Exposure to direct sunlight should beavoided during preparation and administration.

Results and Conclusion

Results through Week 28 for Safety and Efficacy of Intravenous Golimumabin Adult Patients with Active Ankylosing Spondylitis:

Introduction:

GO-ALIVE is a Phase 3, multicenter, randomized, double-blind,placebo-controlled trial designed to evaluate the safety and efficacy ofIV golimumab in adult patients with active AS. Patients (aged ≥18 yrs)had a diagnosis of definite AS (per modified New York criteria) andBASDAI ≥4, total back pain visual analogue scale ≥4, and CRP ≥0.3 mg/dL.Patients were randomized (1:1) to IV golimumab 2 mg/kg at weeks (wks) 0,4, and every 8 wks or placebo at wks 0, 4, and 12, with crossover togolimumab at wk16. Up to 20% of patients could have had a prior anti-TNFagent (other than golimumab), and up to 10% of patients could havecomplete ankylosis of the spine. The primary endpoint was ASAS20 atwk16. Major secondary endpoints were ASAS40, BASDAI50, and change inBASFI score at wk16. Other statistically-controlled assessments wereBASMI, ASAS partial remission, SF-36 PCS/MCS, and ASQoL. Patients weremonitored for adverse and data through wk28 are reported here. Allinvestigators and some sponsor personnel will remain blinded to thetreatment group assignments through the end of the study (wk60); thustreatment group assignments for individual patients are not reportedhere.

Results:

208 patients were randomized and received study agent (placebo: 103;golimumab: 105). Baseline demographic and disease characteristics weresimilar between treatment groups. 78% of patients were male, mean agewas 39 yrs; mean disease duration was 5.5 yrs, 89.9% were HLA-B27positive, 5.8% had complete ankylosis of the spine, 14.4% used a prioranti-TNF. At wk16, significantly greater proportions of golimumabpatients vs placebo had ASAS20 (73.3% vs. 26.2%), ASAS40 (47.6% vs.8.7%), and BASDAI 50 (41.0% vs. 14.6%) responses (all p<0.001; Table).Reductions in BASFI were also significantly greater with golimumab.Improvements in SF-36 PCS/MCS and ASQoL were significantly greater inthe golimumab group vs placebo at wk16. ASAS20 was significantly higherwith golimumab than placebo as early as wk2 (37.1% vs 19.4%; p=0.005).Responses in the golimumab group were maintained through wk28. Placebopatients who crossed over to golimumab at wk16 had improvements inclinical response at wk20 that were maintained through wk28. Throughwk16, 23.3% of placebo patients and 32.4% of golimumab patients had ≥1AE. Infections were the most common AE (placebo, 7.8%; golimumab,11.4%). Through wk28, 34.8% of all golimumab-treated patients had ≥1 AE;nasopharyngitis (5.4%) was the most common. Two platients (1.0%) hadSAEs (pancreatitis, n=1; pneumonia, n=1). There were no opportunisticinfections, malignancies, or deaths through wk28 and the rate ofinfusion reactions was low (1.4%). 3 platients had 4 reactions; nonewere serious or severe.

Conclusion:

IV golimumab 2 mg/kg was efficacious in reducing signs and symptoms ofAS compared with placebo. Golimumab was well-tolerated through wk28 andthe safety profile was consistent with other anti-TNFs, including SCgolimumab.

TABLE 8 Clinical Response Efficacy at week 16. Placebo Golimumab 2 mg/kgPatients randomized, n 103  105 Clinical efficacy ASAS20, n (%) 27(26.2%) 77 (73.3%)** ASAS40, n (%) 9 (8.7%) 50 (47.6%)** BASDAI 50, n(%) 15 (14.6%) 43 (41.0%)** Change from baseline in BASFI n 98 105 mean(SD) −0.5 (2.0) −2.4 (2.1)** ASAS partial remission, n (%) 4 (3.9%) 17(16.2%)* Change from baseline in BASMI (linear) n 96 100 mean (SD) −0.1(0.5) −0.4 (0.6)** Health-related quality of life Change from baselinein SF-36 PCS score n 98 104 mean (SD) 2.9 (6.2) 8.5 (7.5)** Change frombaseline in SF-36 MCS score n 98 104 mean (SD) 0.8 (10.0) 6.5 (9.1)**Change from baseline in ASQoL n 98 104 mean (SD) −1.8 (4.6) −5.4 (5.0)***p < 0.01; **p ≤ 0.001 ASAS20/40, ≥20%/40% improvement in ASsessment inAnkylosing Spondylitis (ASAS) International Working Group criteria;ASQoL, Ankylosing Spondylitis Qualify of Life; BASDAI, Bath AnkylosingSpondylitis Disease Activity Index; BASFI, Bath Ankylosing SpondylitisFunctional Index; BASMI, Bath Ankylosing Spondylitis Metrology Index;SD, standard deviation; SF-36 PCS/MCS, 36-item Short-Form Health SurveyPhysical/Mental Component Summary

TABLE 9 Number of Subjects Who Achieved ASDAS Inactive Disease (<1.3)Through Week 28; Full Analysis Set Placebo Golimumab 2 mg/kg AnalysisSet: Full Analysis Set 103 105 Subjects evaluable for ASDAS 103 105inactive disease (<1.3) at Week 2^(a) Subjects with ASDAS inactive  0 10(9.5%) disease (<1.3) % Difference (95% CI)^(b) 9.6 (3.93, 15.19)p-value^(c) 0.001 Subjects evaluable for ASDAS 103 105 inactive disease(<1.3) at Week 4^(a) Subjects with ASDAS inactive  0 15 (14.3%) disease(<1.3) % Difference (95% CI)^(b) 14.3 (7.60, 21.00) p-value^(c) <0.001Subjects evaluable for ASDAS 103 105 inactive disease (<1.3) at Week8^(a) Subjects with ASDAS inactive 3 (2.9%) 22 (21.0%) disease (<1.3) %Difference (95% CI)^(b) 18.1 (9.66, 26.51) p-value^(c) <0.001 Subjectsevaluable for ASDAS 103 105 inactive disease (<1.3) at Week 12^(a)Subjects with ASDAS inactive 1 (1.0%) 20 (19.0%) disease (<1.3) %Difference (95% CI)^(b) 18.1 (10.34, 25.83) p-value^(c) <0.001 Subjectsevaluable for ASDAS 103 105 inactive disease (<1.3) at Week 16^(a)Subjects with ASDAS inactive 3 (2.9%) 29 (27.6%) disease (<1.3) %Difference (95% CI)^(b) 24.8 (15.62, 33.90) p-value^(c) <0.001 Subjectsevaluable for ASDAS 103 105 inactive disease (<1.3) at Week 20^(a)Subjects with ASDAS inactive 29 (28.2%) 30 (28.6%) disease (<1.3)Subjects evaluable for ASDAS 103 105 inactive disease (<1.3) at Week28^(a) Subjects with ASDAS inactive 24 (23.3%) 31 (29.5%) disease (<1.3)^(a)ASDAS inactive disease (<1.3) is based on imputed data usingtreatment failure (only through Week 16), and LOCF for missing data.^(b)The confidence intervals are based on Wald statistic controlling forprior anti-TNF therapy (Yes, No). ^(c)The p-values are based on CMH testcontrolling for prior anti-TNF therapy (Yes, No).

REFERENCES—Example 9

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1-10. (canceled)
 11. A method for treating a TNF related condition,wherein the TNF related condition is active ankylosing spondylitis, themethod comprising: administering a composition comprising a safe andeffective amount of at least one isolated mammalian anti-TNF antibodycomprising: a. a heavy chain (HC) complementary determining region (CDR)1 comprising an amino acid sequence of SEQ ID NO: 1; b. a HC CDR2comprising an amino acid sequence of SEQ ID NO: 2; wherein position 1 ofSEQ ID NO: 2 is phenylalanine, position 2 of SEQ ID NO: 2 is methionine,position 3 of SEQ ID NO: 2 is serine, position 4 of SEQ ID NO: 2 istyrosine, position 10 of SEQ ID NO: 2 is lysine, position 11 of SEQ IDNO: 2 is tyrosine, and position 17 of SEQ ID NO: 2 is glycine; c. a HCCDR3 comprising an amino acid sequence of SEQ ID NO: 3; wherein position4 of SEQ ID NO: 3 is isoleucine, position 5 of SEQ ID NO: 3 is alanine,and position 9 of SEQ ID NO: 3 is asparagine; d. a light chain (LC) CDR1comprising an amino acid sequence of SEQ ID NO: 4; wherein position 7 ofSEQ ID NO: 4 is tyrosine; e. a LC CDR2 comprising an amino acid sequenceof SEQ ID NO: 5; and f. a LC CDR3 comprising an amino acid sequence ofSEQ ID NO: 6, and at least one pharmaceutically acceptable carrier ordiluent, wherein said composition is administered via IV infusion, andwherein a patient treated with the composition achieves an AnkylosingSpondylitis Disease Activity Score (ASDAS) inactive disease (<1.3) at 4weeks of treatment or at 2 weeks of treatment.
 12. The method accordingto claim 11, wherein said composition is administered such that saidanti-TNF antibody is administered at a dose of 2 mg/kg, administeredover 30±10 minutes, at Weeks 0 and 4, and then every 8 weeks (q8w)thereafter.
 13. The method according to claim 11, further comprisingadministering said composition with or without methotrexate (MTX),sulfasalazine (SSZ) or hydroxychloroquine (HCQ).
 14. A method fortreating a TNF related condition, wherein the TNF related condition isactive ankylosing spondylitis, the method comprising: administering acomposition comprising a safe and effective amount of at least oneisolated mammalian anti-TNF antibody comprising: a. a heavy chain (HC)complementary determining region (CDR) 1 comprising an amino acidsequence of SEQ ID NO: 1; b. a HC CDR2 comprising an amino acid sequenceof SEQ ID NO: 2; wherein position 1 of SEQ ID NO: 2 is phenylalanine,position 2 of SEQ ID NO: 2 is methionine, position 3 of SEQ ID NO: 2 isserine, position 4 of SEQ ID NO: 2 is tyrosine, position 10 of SEQ IDNO: 2 is lysine, position 11 of SEQ ID NO: 2 is tyrosine, and position17 of SEQ ID NO: 2 is glycine; c. a HC CDR3 comprising an amino acidsequence of SEQ ID NO: 3; wherein position 4 of SEQ ID NO: 3 isisoleucine, position 5 of SEQ ID NO: 3 is alanine, and position 9 of SEQID NO: 3 is asparagine; d. a light chain (LC) CDR1 comprising an aminoacid sequence of SEQ ID NO: 4; wherein position 7 of SEQ ID NO: 4 istyrosine; e. a LC CDR2 comprising an amino acid sequence of SEQ ID NO:5; and f. a LC CDR3 comprising an amino acid sequence of SEQ ID NO: 6,and at least one pharmaceutically acceptable carrier or diluent, whereinsaid composition is administered via IV infusion, and wherein at week 16of treatment patients treated with the anti-TNF antibody achieve a meanchange from baseline in one or more criteria selected from the groupconsisting of: Bath Ankylosing Spondylitis Functional Index(BASFI)=−2.4±2.1 Standard Deviation (SD), Bath Ankylosing SpondylitisMetrology Index (BASMI)=−0.4±0.6 SD, 36-item Short-Form Health SurveyPhysical Component Summary (SF-36 PCS)=8.5±7.5 SD, 36-item Short-FormHealth Survey Mental Component Summary (SF-36 MCS)=6.5±9.1 SD, andAnkylosing Spondylitis Qualify of Life questionnaire (ASQoL)=−5.4±5.0SD.
 15. The method according to claim 14, wherein said composition isadministered such that said anti-TNF antibody is administered at a doseof 2 mg/kg, administered over 30±10 minutes, at Weeks 0 and 4, and thenevery 8 weeks (q8w) thereafter.
 16. The method according to claim 14,further comprising administering said composition with or withoutmethotrexate (MTX), sulfasalazine (SSZ) or hydroxychloroquine (HCQ). 17.A method for treating a TNF related condition, wherein the TNF relatedcondition is active ankylosing spondylitis, the method comprising:administering a safe and effective amount of at least one isolatedmammalian anti-TNF antibody comprising: a. a heavy chain (HC)complementary determining region (CDR) 1 comprising an amino acidsequence of SEQ ID NO: 1; b. a HC CDR2 comprising an amino acid sequenceof SEQ ID NO: 2; wherein position 1 of SEQ ID NO: 2 is phenylalanine,position 2 of SEQ ID NO: 2 is methionine, position 3 of SEQ ID NO: 2 isserine, position 4 of SEQ ID NO: 2 is tyrosine, position 10 of SEQ IDNO: 2 is lysine, position 11 of SEQ ID NO: 2 is tyrosine, and position17 of SEQ ID NO: 2 is glycine; c. a HC CDR3 comprising an amino acidsequence of SEQ ID NO: 3; wherein position 4 of SEQ ID NO: 3 isisoleucine, position 5 of SEQ ID NO: 3 is alanine, and position 9 of SEQID NO: 3 is asparagine; d. a light chain (LC) CDR1 comprising an aminoacid sequence of SEQ ID NO: 4; wherein position 7 of SEQ ID NO: 4 istyrosine; e. a LC CDR2 comprising an amino acid sequence of SEQ ID NO:5; and f. a LC CDR3 comprising an amino acid sequence of SEQ ID NO: 6,wherein said anti-TNF antibody is administered via intravenous (IV)infusion, and wherein ≥65% of patients receiving the treatment achieveAssessment in Ankylosing Spondylitis 20 (ASAS20) at week 16 oftreatment.
 18. The method according to claim 17, wherein said ≥65% ofpatients that achieve ASAS20 at week 16 of treatment have a treatmentdifference (improvement compared to placebo) of ≥45%.
 19. The methodaccording to claim 17, wherein said antibody is administered at a doseof 2 mg/kg, administered over 30±10 minutes, at Weeks 0 and 4, and thenevery 8 weeks (q8w) thereafter.
 20. The method according to claim 17,wherein said antibody is administered with or without methotrexate(MTX), sulfasalazine (SSZ) or hydroxychloroquine (HCQ).